Compositions and methods for inhibition of expression of apolipoprotein C-III (APOC3) genes

ABSTRACT

The invention relates to double-stranded ribonucleic acid (dsRNA) targeting an APOC3 gene, and methods of using the dsRNA to inhibit expression of APOC3.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/125,567, filed Dec. 11, 2013, (allowed) which is a National PhaseApplication of International Application No. PCT/US2012/043642, filedJun. 21, 2012, which claims the benefit of U.S. Provisional ApplicationNo. 61/499,620, filed Jun. 23, 2011, all of which are herebyincorporated in their entirety by reference.

REFERENCE TO SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named 33387_US_sequencelisting.txt, created on Month, Mar.3, 2015, with a size of 237,568 bytes. The sequence listing isincorporated by reference.

FIELD OF THE INVENTION

The invention relates to double-stranded ribonucleic acid (dsRNA)targeting an APOC3 gene, and methods of using the dsRNA to inhibitexpression of APOC3.

BACKGROUND OF THE INVENTION

In the U.S., 30% of adults have elevated triglycerides (TG)>150 mg/dL.The prevalence of adults with severe hypertriglyceridemia (TG>500 mg/dL)is 1.7%. Current treatments include lifestyle modification (diet,exercise and smoking cessation), prescription grade fish oil, fibrates,and niacin.

ApoC3 is a secreted liver protein shown to inhibit lipoprotein lipasesthat hydrolyze TG into free fatty acids; inhibit ApoE-mediated hepaticuptake of TG-rich lipoproteins through LDLR and LRP as well as receptorindependent endocytosis; and promote hepatic VLDL secretion. At leastone mutation in the human APOC3 gene has been associated with afavorable lipid profile. (Pollin T I et al. (2008) A null mutation inhuman APOC3 confers a favorable plasma lipid profile and apparentcardioprotection. Science. 322(5908):1702-5).

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.).

SUMMARY OF THE INVENTION

Disclosed herein are double-stranded ribonucleic acid (dsRNA) forinhibiting expression of an APOC3 gene, wherein the dsRNA comprises asense strand and an antisense strand each 30 nucleotides or less inlength, wherein the antisense strand comprises at least 15 contiguousnucleotides of an antisense sequence in Table 1, 2, 6, 7, or 10. In oneembodiment, the dsRNA comprises a sense strand consisting of thenucleotide sequence SEQ ID NO:70 and an antisense strand consisting of anucleotide sequence SEQ ID NO: 151 (AD-45149.1UM). In anotherembodiment, the sense strand sequence is selected from Table 1, 2, 6, 7,or 10, and the antisense strand is selected from Table 1, 2, 6, 7, or10.

In some embodiments, at least one nucleotide of the dsRNA is a modifiednucleotide, e.g., at least one modified nucleotide is chosen from thegroup consisting of: a 2′-O-methyl modified nucleotide, a nucleotidecomprising a 5′-phosphorothioate group, and a terminal nucleotide linkedto a cholesteryl derivative or dodecanoic acid bisdecylamide group, a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide.

In some embodiments, at least one strand comprises a 3′ overhang of atleast 1 nucleotide or each strand comprises a 3′ overhang of at 2nucleotides.

Any dsRNA of the invention can further comprising a ligand, for examplea ligand that is conjugated to the 3′ end of the sense strand of thedsRNA. In some embodiments, a dsRNA of the invention further comprisesat least one N-Acetyl-Galactosamine.

In addition the invention provides a cell comprising any dsRNA of theinvention; a vector encoding at least one strand of any dsRNA of theinvention and a cell comprising the vector.

Also included in the invention are pharmaceutical compositions forinhibiting expression of an APOC3 gene comprising any dsRNA of theinvention. The pharmaceutical composition can include a lipidformulation, e.g., a lipid formulation comprising MC3.

Another aspect of the invention is a method of inhibiting APOC3expression in a cell, the method comprising: (a) contacting the cell aAPOC3 dsRNA of the invention and (b) maintaining the cell produced instep (a) for a time sufficient to obtain degradation of the mRNAtranscript of an APOC3 gene, thereby inhibiting expression of the APOC3gene in the cell. In some embodiments APOC3 expression is inhibited byat least 30%.

A further aspect of the invention is a method of treating a disordermediated by APOC3 expression comprising administering to a human in needof such treatment a therapeutically effective amount of the APOC3 dsRNAof the invention or a pharmaceutical composition of the invention. Thedisorder can be, e.g., elevated triglyceride levels, e.g., triglyceridelevels>150 mg/dL or >500 mg/dL. In some embodiments administrationcauses an increase in lipoprotein lipase and/or hepatic lipase activity.The dsRNA or the pharmaceutical composition can be administered at adose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about50 mg/kg.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect on target mRNA, triglyceride (TG)and total cholesterol levels in mice after treatment with siRNAtargeting APOC3 (“siRNA#1” and siRNA#2”).

FIG. 2 shows the structure of GalNAc.

FIG. 3 shows the structure of an siRNA conjugated to Chol-p-(GalNAc)₃via phosphate linkage at the 3′ end.

FIG. 4 shows the structure of an siRNA conjugated to LCO(GalNAc)₃ (a(GalNAc)₃-3′-Lithocholic-oleoyl siRNA Conjugate).

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

The invention provides dsRNAs and methods of using the dsRNAs forinhibiting the expression of an APOC3 gene in a cell or a mammal wherethe dsRNA targets an APOC3 gene. The invention also providescompositions and methods for treating pathological conditions anddiseases in a mammal caused by the expression of an APOC3 gene. AN APOC3dsRNA directs the sequence-specific degradation of APOC3 mRNA.

DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dT” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine. However, it will be understood that the term“ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also referto a modified nucleotide, as further detailed below, or a surrogatereplacement moiety. The skilled person is well aware that guanine,cytosine, adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of the invention by a nucleotide containing,for example, inosine. Sequences comprising such replacement moieties areembodiments of the invention.

“APOC3” refers to the Apolipoprotein C-III gene. According to the NCBINLM website, Apolipoprotein C-III is a very low density lipoprotein(VLDL) protein. APOC3 inhibits lipoprotein lipase and hepatic lipase; itis thought to delay catabolism of triglyceride-rich particles. TheAPOA1, APOC3 and APOA4 genes are closely linked in both rat and humangenomes. The A-I and A-IV genes are transcribed from the same strand,while the A-1 and C-III genes are convergently transcribed. An increasein apoC-III levels induces the development of hypertriglyceridemia. Ahuman APOC3 mRNA sequence is GenBank accession number NM_000040.1,included herein as SEQ ID NO: 1. A cynomolgus monkey (Macacafascicularis) ANGPTL3 mRNA sequence is GenBank accession numberX68359.1.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an APOC3 gene, including mRNA that is a product of RNA processing ofa primary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.

For example, a first nucleotide sequence can be described ascomplementary to a second nucleotide sequence when the two sequenceshybridize (e.g., anneal) under stringent hybridization conditions.Hybridization conditions include temperature, ionic strength, pH, andorganic solvent concentration for the annealing and/or washing steps.The term stringent hybridization conditions refers to conditions underwhich a first nucleotide sequence will hybridize preferentially to itstarget sequence, e.g., a second nucleotide sequence, and to a lesserextent to, or not at all to, other sequences. Stringent hybridizationconditions are sequence dependent, and are different under differentenvironmental parameters. Generally, stringent hybridization conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the nucleotide sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the first nucleotide sequences hybridize to a perfectlymatched target sequence. An extensive guide to the hybridization ofnucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I, chap. 2, “Overview of principles of hybridization and thestrategy of nucleic acid probe assays,” Elsevier, N.Y. (“Tijssen”).

Other conditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding APOC3) including a 5′ UTR, an openreading frame (ORF), or a 3′ UTR. For example, a polynucleotide iscomplementary to at least a part of an APOC3 mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding APOC3.

In one embodiment, the antisense strand of the dsRNA is sufficientlycomplementary to a target mRNA so as to cause cleavage of the targetmRNA.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. In general, the majority of nucleotides ofeach strand are ribonucleotides, but as described in detail herein, eachor both strands can also include at least one non-ribonucleotide, e.g.,a deoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, “dsRNA” may include chemical modifications toribonucleotides, including substantial modifications at multiplenucleotides and including all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an siRNA typemolecule, are encompassed by “dsRNA” for the purposes of thisspecification and claims.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” Where the two strands are connected covalently by means otherthan an uninterrupted chain of nucleotides between the 3′-end of onestrand and the 5′-end of the respective other strand forming the duplexstructure, the connecting structure is referred to as a “linker.” TheRNA strands may have the same or a different number of nucleotides. Themaximum number of base pairs is the number of nucleotides in theshortest strand of the dsRNA minus any overhangs that are present in theduplex. In addition to the duplex structure, a dsRNA may comprise one ormore nucleotide overhangs. The term “siRNA” is also used herein to referto a dsRNA as described above.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

As used herein, the term “nucleic acid lipid particle” includes the term“SNALP” and refers to a vesicle of lipids coating a reduced aqueousinterior comprising a nucleic acid such as a dsRNA or a plasmid fromwhich a dsRNA is transcribed. Nucleic acid lipid particles, e.g., SNALPare described, e.g., in U.S. Patent Application Publication Nos.20060240093, 20070135372, and U.S. Ser. No. 61/045,228 filed on Apr. 15,2008. These applications are hereby incorporated by reference.

“Introducing into a cell,” when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell,” wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection. Further approaches are describedherein or known in the art.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of” and the like in as far asthey refer to an APOC3 gene, herein refer to the at least partialsuppression of the expression of an APOC3 gene, as manifested by areduction of the amount of mRNA which may be isolated from a first cellor group of cells in which an APOC3 gene is transcribed and which has orhave been treated such that the expression of an APOC3 gene isinhibited, as compared to a second cell or group of cells substantiallyidentical to the first cell or group of cells but which has or have notbeen so treated (control cells). The degree of inhibition is usuallyexpressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to APOC3 geneexpression, e.g., the amount of protein encoded by an APOC3 gene whichis secreted by a cell, or the number of cells displaying a certainphenotype, e.g., apoptosis. In principle, APOC3 gene silencing may bedetermined in any cell expressing the target, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given dsRNA inhibitsthe expression of an APOC3 gene by a certain degree and therefore isencompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

For example, in certain instances, expression of an APOC3 gene issuppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% by administration of the double-stranded oligonucleotide featuredin the invention. In some embodiments, an APOC3 gene is suppressed by atleast about 60%, 70%, or 80% by administration of the double-strandedoligonucleotide featured in the invention. In some embodiments, an APOC3gene is suppressed by at least about 85%, 90%, or 95% by administrationof the double-stranded oligonucleotide featured in the invention.

As used herein in the context of APOC3 expression, the terms “treat,”“treatment,” and the like, refer to relief from or alleviation ofpathological processes mediated by APOC3 expression. In the context ofthe present invention insofar as it relates to any of the otherconditions recited herein below (other than pathological processesmediated by APOC3 expression), the terms “treat,” “treatment,” and thelike mean to relieve or alleviate at least one symptom associated withsuch condition, or to slow or reverse the progression of such condition.

As used herein, the phrases “effective amount” refers to an amount thatprovides a therapeutic benefit in the treatment, prevention, ormanagement of pathological processes mediated by APOC3 expression or anovert symptom of pathological processes mediated by APOC3 expression.The specific amount that is effective can be readily determined by anordinary medical practitioner, and may vary depending on factors knownin the art, such as, for example, the type of pathological processesmediated by APOC3 expression, the patient's history and age, the stageof pathological processes mediated by APOC3 expression, and theadministration of other anti-pathological processes mediated by APOC3expression agents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter. For example, a therapeuticallyeffective amount of a dsRNA targeting APOC3 can reduce APOC3 serumlevels by at least 25%.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

Double-Stranded Ribonucleic Acid (dsRNA)

As described in more detail herein, the invention providesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of an APOC3 gene in a cell or mammal, where the dsRNAincludes an antisense strand having a region of complementarity which iscomplementary to at least a part of an mRNA formed in the expression ofan APOC3 gene, and where the region of complementarity is less than 30nucleotides in length, generally 19-24 nucleotides in length, and wheresaid dsRNA, upon contact with a cell expressing said APOC3 gene,inhibits the expression of said APOC3 gene by at least 30% as assayedby, for example, a PCR or branched DNA (bDNA)-based method, or by aprotein-based method, such as by Western blot. Expression of an APOC3gene can be reduced by at least 30% when measured by an assay asdescribed in the Examples below. For example, expression of an APOC3gene in cell culture, such as in Hep3B cells, can be assayed bymeasuring APOC3 mRNA levels, such as by bDNA or TaqMan assay, or bymeasuring protein levels, such as by ELISA assay. The dsRNA of theinvention can further include one or more single-stranded nucleotideoverhangs.

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc. The dsRNA includes two RNA strands that aresufficiently complementary to hybridize to form a duplex structure. Onestrand of the dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence, derived from the sequence of anmRNA formed during the expression of an APOC3 gene, the other strand(the sense strand) includes a region that is complementary to theantisense strand, such that the two strands hybridize and form a duplexstructure when combined under suitable conditions. Generally, the duplexstructure is between 15 and 30 or between 25 and 30, or between 18 and25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 basepairs in length. In one embodiment the duplex is 19 base pairs inlength. In another embodiment the duplex is 21 base pairs in length.When two different siRNAs are used in combination, the duplex lengthscan be identical or can differ.

Each strand of the dsRNA of invention is generally between 15 and 30, orbetween 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides inlength. In other embodiments, each is strand is 25-30 nucleotides inlength. Each strand of the duplex can be the same length or of differentlengths. When two different siRNAs are used in combination, the lengthsof each strand of each siRNA can be identical or can differ.

The dsRNA of the invention include dsRNA that are longer than 21-23nucleotides, e.g., dsRNA that are long enough to be processed by theRNase III enzyme Dicer into 21-23 basepair siRNA which are thenincorporated into a RISC. Accordingly, a dsRNA of the invention can beat least 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or at least 100basepairs in length.

The dsRNA of the invention can include one or more single-strandedoverhang(s) of one or more nucleotides. In one embodiment, at least oneend of the dsRNA has a single-stranded nucleotide overhang of 1 to 4,generally 1 or 2 nucleotides. In another embodiment, the antisensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the sense strand. In further embodiments, the sensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the antisense strand.

A dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties than the blunt-ended counterpart. In someembodiments the presence of only one nucleotide overhang strengthens theinterference activity of the dsRNA, without affecting its overallstability. A dsRNA having only one overhang has proven particularlystable and effective in vivo, as well as in a variety of cells, cellculture mediums, blood, and serum. Generally, the single-strandedoverhang is located at the 3′-terminal end of the antisense strand or,alternatively, at the 3′-terminal end of the sense strand. The dsRNA canalso have a blunt end, generally located at the 5′-end of the antisensestrand. Such dsRNAs can have improved stability and inhibitory activity,thus allowing administration at low dosages, i.e., less than 5 mg/kgbody weight of the recipient per day. Generally, the antisense strand ofthe dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

In one embodiment, an APOC3 gene is a human APOC3 gene. In specificembodiments, the sense strand of the dsRNA is one of the sense sequencesfrom Tables 1, 2, 6, 7, 11 or 12, and the antisense strand is one of theantisense sequences of Tables 1, 2, 6, 7, 11 or 12. Alternativeantisense agents that target elsewhere in the target sequence providedin Tables 1, 2, 6, 7, 11 or 12 can readily be determined using thetarget sequence and the flanking APOC3 sequence.

The skilled person is well aware that dsRNAs having a duplex structureof between 20 and 23, but specifically 21, base pairs have been hailedas particularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger dsRNAs can be effective as well. In the embodiments describedabove, by virtue of the nature of the oligonucleotide sequences providedin Tables 1, 2, 6, 7, 11 or 12, the dsRNAs featured in the invention caninclude at least one strand of a length described herein. It can bereasonably expected that shorter dsRNAs having one of the sequences ofTables 1, 2, 6, 7, 11 or 12 minus only a few nucleotides on one or bothends may be similarly effective as compared to the dsRNAs describedabove. Hence, dsRNAs having a partial sequence of at least 15, 16, 17,18, 19, 20, or more contiguous nucleotides from one of the sequences ofTables 1, 2, 6, 7, 11 or 12, and differing in their ability to inhibitthe expression of an APOC3 gene in an assay as described herein below bynot more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNAcomprising the full sequence, are contemplated by the invention.Further, dsRNAs that cleave within a desired APOC3 target sequence canreadily be made using the corresponding APOC3 antisense sequence and acomplementary sense sequence.

In addition, the dsRNAs provided in Tables 1, 2, 6, 7, 11 or 12 identifya site in an APOC3 that is susceptible to RNAi based cleavage. As such,the present invention further features dsRNAs that target within thesequence targeted by one of the agents of the present invention. As usedherein, a second dsRNA is said to target within the sequence of a firstdsRNA if the second dsRNA cleaves the message anywhere within the mRNAthat is complementary to the antisense strand of the first dsRNA. Such asecond dsRNA will generally consist of at least 15 contiguousnucleotides from one of the sequences provided in Tables 1, 2, 6, 7, 11or 12 coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in an APOC3 gene.

Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art. The cleavage site on the target mRNA of adsRNA can be determined using methods generally known to one of ordinaryskill in the art, e.g., the 5′-RACE method described in Soutschek etal., Nature; 2004, Vol. 432, pp. 173-178 (which is herein incorporatedby reference for all purposes).

The dsRNA featured in the invention can contain one or more mismatchesto the target sequence. In one embodiment, the dsRNA featured in theinvention contains no more than 3 mismatches. If the antisense strand ofthe dsRNA contains mismatches to a target sequence, it is preferablethat the area of mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of an APOC3 gene, the dsRNA generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed within the invention can be used to determine whether a dsRNAcontaining a mismatch to a target sequence is effective in inhibitingthe expression of an APOC3 gene. Consideration of the efficacy of dsRNAswith mismatches in inhibiting expression of an APOC3 gene is important,especially if the particular region of complementarity in an APOC3 geneis known to have polymorphic sequence variation within the population.

In another aspect, the invention is a single-stranded antisenseoligonucleotide RNAi. An antisense oligonucleotide is a single-strandedoligonucleotide that is complementary to a sequence within the targetmRNA. Antisense oligonucleotides can inhibit translation in astoichiometric manner by base pairing to the mRNA and physicallyobstructing the translation machinery, see Dias, N. et al., (2002) Mol.Cancer Ther. 1:347-355. Antisense oligonucleotides can also inhibittarget protein expression by binding to the mRNA target and promotingmRNA target destruction via RNase-H. The single-stranded antisense RNAmolecule can be about 13 to about 30 nucleotides in length and have asequence that is complementary to a target sequence. For example, thesingle-stranded antisense RNA molecule can comprise a sequence that isat least about 13, 14, 15, 16, 17, 18, 19, 20, or more contiguousnucleotides from one of the antisense sequences in Table 1, 2, 6, 7, or10.

Modifications

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids featured in the invention may besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S. L. et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Specific examplesof dsRNA compounds useful in this invention include dsRNAs containingmodified backbones or no natural internucleoside linkages. As defined inthis specification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Modified dsRNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Modified dsRNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or ore or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other suitable dsRNA mimetics, both the sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,a dsRNA mimetic that has been shown to have excellent hybridizationproperties, is referred to as a peptide nucleic acid (PNA). In PNAcompounds, the sugar backbone of a dsRNA is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representative U.S.patents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Further teaching of PNAcompounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Other embodiments of the invention are dsRNAs with phosphorothioatebackbones and oligonucleosides with heteroatom backbones, and inparticular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene(methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Preferred dsRNAs comprise one of the following at the 2′ position: OH;F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred dsRNAs comprise one of the following at the 2′ position:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of a dsRNA, or a group for improving thepharmacodynamic properties of a dsRNA, and other substituents havingsimilar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxygroup. A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other preferred modifications include 2′-methoxy (2′-OCH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on the dsRNA,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.DsRNAs may also have sugar mimetics such as cyclobutyl moieties in placeof the pentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

dsRNAs may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Conjugates

Another modification of the dsRNAs of the invention involves chemicallylinking to the dsRNA one or more moieties or conjugates which enhancethe activity, cellular distribution or cellular uptake of the dsRNA.Such moieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989,86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let.,1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan etal., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg.Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuket al., Biochimie, 1993, 75:49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within a dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a dsRNA compound. These dsRNAs typically contain at leastone region wherein the dsRNA is modified so as to confer upon the dsRNAincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of dsRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter dsRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxy dsRNAs hybridizing to the same target region.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such dsRNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of dsRNAs bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the dsRNA still bound to the solid support orfollowing cleavage of the dsRNA in solution phase. Purification of thedsRNA conjugate by HPLC typically affords the pure conjugate.

Conjugating a ligand to a dsRNA can enhance its cellular absorption aswell as targeting to a particular tissue or uptake by specific types ofcells such as liver cells. In certain instances, a hydrophobic ligand isconjugated to the dsRNA to facilitate direct permeation of the cellularmembrane and or uptake across the liver cells. Alternatively, the ligandconjugated to the dsRNA is a substrate for receptor-mediatedendocytosis. These approaches have been used to facilitate cellpermeation of antisense oligonucleotides as well as dsRNA agents. Forexample, cholesterol has been conjugated to various antisenseoligonucleotides resulting in compounds that are substantially moreactive compared to their non-conjugated analogs. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103. Otherlipophilic compounds that have been conjugated to oligonucleotidesinclude 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, andmenthol. One example of a ligand for receptor-mediated endocytosis isfolic acid. Folic acid enters the cell by folate-receptor-mediatedendocytosis. dsRNA compounds bearing folic acid would be efficientlytransported into the cell via the folate-receptor-mediated endocytosis.Li and coworkers report that attachment of folic acid to the 3′-terminusof an oligonucleotide resulted in an 8-fold increase in cellular uptakeof the oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res.1998, 15, 1540. Other ligands that have been conjugated tooligonucleotides include polyethylene glycols, carbohydrate clusters,cross-linking agents, porphyrin conjugates, delivery peptides and lipidssuch as cholesterol and cholesterylamine. Examples of carbohydrateclusters include Chol-p-(GalNAc)₃ (N-acetyl galactosamine cholesterol)and LCO(GalNAc)₃ (N-acetyl galactosamine-3′-Lithocholic-oleoyl.

Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, adsRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated dsRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In one embodiment, themonosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

Linkers

In some embodiments, the conjugate or ligand described herein can beattached to a dsRNA of the invention with various linkers that can becleavable or non cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular dsRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)— (SEQ ID NO: 13), where RA and RB are the R groupsof the two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above.

In one embodiment, a dsRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of dsRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more GalNAc (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXI)-(XXXIV):

wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C representindependently for each occurrence 0-20 and wherein the repeating unitcan be the same or different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O.

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5c) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXV):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II_VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

Vector Encoded dsRNAs

In another aspect, APOC3 dsRNA molecules are expressed fromtranscription units inserted into DNA or RNA vectors (see, e.g.,Couture, A, et al., TIG. (1996), 12:5-10; Skillem, A., et al.,International PCT Publication No. WO 00/22113, Conrad, International PCTPublication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Thesetransgenes can be introduced as a linear construct, a circular plasmid,or a viral vector, which can be incorporated and inherited as atransgene integrated into the host genome. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. Inone embodiment, a dsRNA is expressed as an inverted repeat joined by alinker polynucleotide sequence such that the dsRNA has a stem and loopstructure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Cornette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g.,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors featured in the invention can bepseudotyped with surface proteins from vesicular stomatitis virus (VSV),rabies, Ebola, Mokola, and the like. AAV vectors featured in theinvention can be made to target different cells by engineering thevectors to express different capsid protein serotypes. For example, anAAV vector expressing a serotype 2 capsid on a serotype 2 genome iscalled AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can bereplaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.Techniques for constructing AAV vectors which express different capsidprotein serotypes are within the skill in the art; see, e.g., RabinowitzJ E et al. (2002), J Virol 76:791-801, the entire disclosure of which isherein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Domburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Viral vectors can be derived from AV and AAV. In one embodiment, thedsRNA featured in the invention is expressed as two separate,complementary single-stranded RNA molecules from a recombinant AAVvector having, for example, either the U6 or H1 RNA promoters, or thecytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA featured in the invention,a method for constructing the recombinant AV vector, and a method fordelivering the vector into target cells, are described in Xia H et al.(2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA featured in the invention,methods for constructing the recombinant AV vector, and methods fordelivering the vectors into target cells are described in Samulski R etal. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol,70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are herein incorporated byreference.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector featured in the invention may be a eukaryotic RNA polymerase I(e.g., ribosomal RNA promoter), RNA polymerase II (e.g., CMV earlypromoter or actin promoter or U1 snRNA promoter) or generally RNApolymerase III promoter (e.g., U6 snRNA or 7SK RNA promoter) or aprokaryotic promoter, for example the T7 promoter, provided theexpression plasmid also encodes T7 RNA polymerase required fortranscription from a T7 promoter. The promoter can also direct transgeneexpression to the pancreas (see, e.g., the insulin regulatory sequencefor pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g., Oligofectamine)or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiplelipid transfections for dsRNA-mediated knockdowns targeting differentregions of a single APOC3 gene or multiple APOC3 genes over a period ofa week or more are also contemplated by the invention. Successfulintroduction of vectors into host cells can be monitored using variousknown methods. For example, transient transfection can be signaled witha reporter, such as a fluorescent marker, such as Green FluorescentProtein (GFP). Stable transfection of cells ex vivo can be ensured usingmarkers that provide the transfected cell with resistance to specificenvironmental factors (e.g., antibiotics and drugs), such as hygromycinB resistance.

APOC3 specific dsRNA molecules can also be inserted into vectors andused as gene therapy vectors for human patients. Gene therapy vectorscan be delivered to a subject by, for example, intravenous injection,local administration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or caninclude a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

Pharmaceutical Compositions Containing dsRNA

In one embodiment, the invention provides pharmaceutical compositionscontaining a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition containing the dsRNAis useful for treating a disease or disorder associated with theexpression or activity of an APOC3 gene, such as pathological processesmediated by APOC3 expression. Such pharmaceutical compositions areformulated based on the mode of delivery.

The pharmaceutical compositions featured herein are administered indosages sufficient to inhibit expression of an APOC3 gene.

In general, a suitable dose of dsRNA will be in the range of 0.01 to200.0 milligrams per kilogram body weight of the recipient per day,generally in the range of 1 to 50 mg per kilogram body weight per day.

Subjects can be administered a therapeutic amount of dsRNA, such asabout 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, 0.5mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg,1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kg dsRNA, 2.9mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kg dsRNA, 3.3mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kg dsRNA, 3.7mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kg dsRNA, 4.1mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kg dsRNA, 4.5mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kg dsRNA, 4.9mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kg dsRNA, 5.3mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kg dsRNA, 5.7mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kg dsRNA, 6.1mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kg dsRNA, 6.5mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kg dsRNA, 6.9mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kg dsRNA, 7.3mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kg dsRNA, 7.7mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kg dsRNA, 8.1mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4 mg/kg dsRNA, 8.5mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kg dsRNA, 8.9mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kg dsRNA, 9.3mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kg dsRNA, 9.7mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 10 mg/kgdsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA, 30 mg/kg dsRNA,35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about 50 mg/kg dsRNA.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

The pharmaceutical composition may be administered once daily or thedsRNA may be administered as two, three, or more sub-doses atappropriate intervals throughout the day or even using continuousinfusion or delivery through a controlled release formulation. In thatcase, the dsRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage. The dosage unit canalso be compounded for delivery over several days, e.g., using aconventional sustained release formulation which provides sustainedrelease of the dsRNA over a several day period. Sustained releaseformulations are well known in the art and are particularly useful fordelivery of agents at a particular site, such as could be used with theagents of the present invention. In this embodiment, the dosage unitcontains a corresponding multiple of the daily dose.

The effect of a single dose on APOC3 levels is long lasting, such thatsubsequent doses are administered at not more than 3, 4, or 5 dayintervals, or at not more than 1, 2, 3, or 4 week intervals, or at notmore than 5, 6, 7, 8, 9, or 10 week intervals.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by APOC3 expression. Such models are used for in vivo testingof dsRNA, as well as for determining a therapeutically effective dose. Asuitable mouse model is, for example, a mouse containing a plasmidexpressing human APOC3. Another suitable mouse model is a transgenicmouse carrying a transgene that expresses human APOC3.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

The dsRNAs featured in the invention can be administered in combinationwith other known agents effective in treatment of pathological processesmediated by target gene expression. In any event, the administeringphysician can adjust the amount and timing of dsRNA administration onthe basis of results observed using standard measures of efficacy knownin the art or described herein.

Administration

The present invention also includes pharmaceutical compositions andformulations which include the dsRNA compounds featured in theinvention. The pharmaceutical compositions of the present invention maybe administered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including buccal and sublingual),pulmonary, e.g., by inhalation or insufflation of powders or aerosols,including by nebulizer; intratracheal, intranasal, epidermal andtransdermal, oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g.,intraparenchymal, intrathecal or intraventricular administration.

The dsRNA can be delivered in a manner to target a particular tissue.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Suitable topical formulations include those inwhich the dsRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearoylphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively, dsRNAs maybe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, an APOC3 dsRNA featured in the invention is fullyencapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP,SNALP, or other nucleic acid-lipid particle. As used herein, the term“SNALP” refers to a stable nucleic acid-lipid particle, including SPLP.As used herein, the term “SPLP” refers to a nucleic acid-lipid particlecomprising plasmid DNA encapsulated within a lipid vesicle. SNALPs andSPLPs typically contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). SNALPs and SPLPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). SPLPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The particlesof the present invention typically have a mean diameter of about 50 nmto about 150 nm, more typically about 60 nm to about 130 nm, moretypically about 70 nm to about 110 nm, most typically about 70 nm toabout 90 nm, and are substantially nontoxic. In addition, the nucleicacids when present in the nucleic acid-lipid particles of the presentinvention are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. In some embodiments the lipid to dsRNA ratio can be about1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 11:1.

In general, the lipid-nucleic acid particle is suspended in a buffer,e.g., PBS, for administration. In one embodiment, the pH of the lipidformulated siRNA is between 6.8 and 7.8, e.g., 7.3 or 7.4. Theosmolality can be, e.g., between 250 and 350 mOsm/kg, e.g., around 300,e.g., 298, 299, 300, 301, 302, 303, 304, or 305.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(C12-200 or Tech G1), or a mixture thereof. The cationic lipid maycomprise from about 20 mol % to about 50 mol % or about 40 mol % of thetotal lipid present in the particle.

The non-cationic lipid may be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid may be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Cl₆), or aPEG-distearyloxypropyl (C₁₈). Other examples of PEG conjugates includePEG-cDMA (N-[(methoxy poly(ethyleneglycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine), mPEG2000-DMG(mPEG-dimyrystylglycerol (with an average molecular weight of 2,000) andPEG-C-DOMG (R-3-[(ω-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxlpropyl-3-amine). The conjugatedlipid that prevents aggregation of particles may be from 0 mol % toabout 20 mol % or about 1.0, 1.1., 1.2, 0.13, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, or 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

For example, the lipid-siRNA particle can include 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

In still another embodiment, the compound1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1) can be used to prepare lipid-siRNA particles. For example, thedsRNA can be formulated in a lipid formulation comprising Tech-G1,distearoyl phosphatidylcholine (DSPC), cholesterol and mPEG2000-DMG at amolar ratio of 50:10:38.5:1.5 at a total lipid to siRNA ratio of 7:1(wt:wt).

In one embodiment, the lipidoid ND98.4HCl (MW 1487), Cholesterol(Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be usedto prepare lipid-siRNA nanoparticles (i.e., LNP01 particles). LNP01formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary formulations are described in Table A.

TABLE A cationic lipid/non-cationic lipid/ cholesterol/PEG-lipidconjugate Cationic Mol % ratios Lipid Lipid:siRNA ratio SNALP DLinDMADLinDMA/DPPC/Cholesterol/PEG-cDMA (57.1/7.1/34.4/1.4) lipid:siRNA ~7:1S-XTC XTC XTC/DPPC/Cholesterol/PEG-cDMA 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP05 XTC XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5lipid:siRNA ~6:1 LNP06 XTC XTC/DSPC/Cholesterol/PEG-DMG57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP07 XTCXTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP08 XTCXTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP09 XTCXTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10ALN100 ALN100/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1LNP11 MC3 MC-3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1LNP12 C12-200 C12-200/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5Lipid:siRNA 10:1 LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG50/10/38.5/1.5 Lipid:siRNA: 10:1

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. filed Jun. 10,2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S.Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and InternationalApplication No. PCT/US2010/022614, filed Jan. 29, 2010, which are herebyincorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No.61/185,800, filed Jun. 10, 2009, and International Application No.PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated byreference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totalsiRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated siRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” siRNA content (as measured by thesignal in the absence of surfactant) from the total siRNA content.Percent entrapped siRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention may be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. dsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pullulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyomithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Pub. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 2, p. 335; Higuchi et al., in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions areoften biphasic systems comprising two immiscible liquid phasesintimately mixed and dispersed with each other. In general, emulsionsmay be of either the water-in-oil (w/o) or the oil-in-water (o/w)variety. When an aqueous phase is finely divided into and dispersed asminute droplets into a bulk oily phase, the resulting composition iscalled a water-in-oil (w/o) emulsion. Alternatively, when an oily phaseis finely divided into and dispersed as minute droplets into a bulkaqueous phase, the resulting composition is called an oil-in-water (o/w)emulsion. Emulsions may contain additional components in addition to thedispersed phases, and the active drug which may be present as a solutionin either the aqueous phase, oily phase or itself as a separate phase.Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, andanti-oxidants may also be present in emulsions as needed. Pharmaceuticalemulsions may also be multiple emulsions that are comprised of more thantwo phases such as, for example, in the case of oil-in-water-in-oil(o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complexformulations often provide certain advantages that simple binaryemulsions do not. Multiple emulsions in which individual oil droplets ofan o/w emulsion enclose small water droplets constitute a w/o/wemulsion. Likewise a system of oil droplets enclosed in globules ofwater stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, non-swelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of ease of formulation, as well as efficacyfrom an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions of dsRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the dsRNAs and nucleicacids of the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly dsRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of dsRNAs through the mucosa isenhanced. In addition to bile salts and fatty acids, these penetrationenhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C.sub. 1-10 alkyl esters thereof (e.g., methyl, isopropyland t-butyl), and mono- and di-glycerides thereof (i.e., oleate,laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44,651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. Suitable bile salts include, forexample, cholic acid (or its pharmaceutically acceptable sodium salt,sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholicacid (sodium deoxycholate), glucholic acid (sodium glucholate),glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of dsRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Suitable chelating agents include butare not limited to disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines)(Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33;Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of dsRNAs throughthe alimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The co-administration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it isco-administered with polyinosinic acid, dextran sulfate, polycytidicacid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyaoet al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA &Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pre-gelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more dsRNA compounds and (b) one or moreanti-cytokine biologic agents which function by a non-RNAi mechanism.Examples of such biologics include, biologics that target IL1β (e.g.,anakinra), IL6 (tocilizumab), or TNF (etanercept, infliximab, adlimumab,or certolizumab).

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the dsRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby APOC3 expression. In any event, the administering physician canadjust the amount and timing of dsRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

Methods for Inhibiting Expression of an APOC3 Gene

The present invention also provides methods of using a dsRNA of theinvention and/or a composition containing an iRNA of the invention toreduce and/or inhibit APOC3 expression in a cell. The methods includecontacting the cell with a dsRNA of the invention and maintaining thecell for a time sufficient to obtain degradation of the mRNA transcriptof an APOC3 gene, thereby inhibiting expression of the APOC3 gene in thecell. Reduction in gene expression can be assessed by any methods knownin the art. For example, a reduction in the expression of APOC3 may bedetermined by determining the mRNA expression level of APOC3 usingmethods routine to one of ordinary skill in the art, e.g., Northernblotting, qRT-PCR, by determining the protein level of APOC3 usingmethods routine to one of ordinary skill in the art, such as Westernblotting, immunological techniques, and/or by determining a biologicalactivity of APOC3, such as affecting one or more molecules associatedwith triglyceride levels, e.g., lipoproteinlipase (LPL) and/or hepaticlipase, or in an in vivo setting, a triglyceride level itself.

In the methods of the invention the cell may be contacted in vitro or invivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses an APOC3 gene. A cell suitable for use in themethods of the invention may be a mammalian cell, e.g., a primate cell(such as a human cell or a non-human primate cell, e.g., a monkey cellor a chimpanzee cell), a non-primate cell (such as a cow cell, a pigcell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbitcell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dogcell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell,or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), ora whale cell. In one embodiment, the cell is a human cell, e.g., a humanliver cell.

APOC3 expression is inhibited in the cell by at least about 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,or about 100%.

The in vivo methods of the invention may include administering to asubject a composition containing a dsRNA, where the dsRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the APOC3 gene of the mammal to be treated. When theorganism to be treated is a mammal such as a human, the composition canbe administered by any means known in the art including, but not limitedto oral, intraperitoneal, or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal), intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by intravenousinfusion or injection or subcutaneous injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the dsRNA in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof APOC3, or a therapeutic or prophylactic effect. A depot injection mayalso provide more consistent serum concentrations. Depot injections mayinclude subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the dsRNA to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of an APOC3gene in a mammal. The methodsinclude administering to the mammal a composition comprising a dsRNAthat targets an APOC3 gene in a cell of the mammal and maintaining themammal for a time sufficient to obtain degradation of the mRNAtranscript of the APOC3 gene, thereby inhibiting expression of the APOC3gene in the cell. Reduction in gene expression can be assessed by anymethods known it the art and by methods, e.g. qRT-PCR, described herein.Reduction in protein production can be assessed by any methods known itthe art and by methods, e.g. ELISA, described herein. In one embodiment,a puncture liver biopsy sample serves as the tissue material formonitoring the reduction in APOC3 gene and/or protein expression. Inother embodiments, inhibition of the expression of an APOC3 gene ismonitored indirectly by, for example, determining the expression and/oractivity of a gene in an APOC3 pathway. For example, the activity oflipoprotein lipase (LPL) or hepatic lipase can be monitored to determinethe inhibition of expression of an APOC3 gene. Triglyceride levels in asample, e.g., a blood or liver sample, may also be measured. Inhibitionof APOC3 inhibition can also be monitored by observing the effect onclinical presentations of elevated triglyceride levels, e.g., the effecton premature chronic heart disease (CHD), eruptive xanthoma,hepatosplenomegaly, and pancreatitis. Suitable assays are furtherdescribed in the Examples section below.

The present invention further provides methods of treatment of a subjectin need thereof. The treatment methods of the invention includeadministering a dsRNA of the invention to a subject, e.g., a subjectthat would benefit from a reduction and/or inhibition of APOC3expression in a therapeutically effective amount of a dsRNA targeting anAPOC3 gene or a pharmaceutical composition comprising a dsRNA targetingan APOC3 gene.

A dsRNA of the invention may be administered in “naked” form, or as a“free dsRNA.” A naked dsRNA is administered in the absence of apharmaceutical composition. The naked dsRNA may be in a suitable buffersolution. The buffer solution may comprise acetate, citrate, prolamine,carbonate, or phosphate, or any combination thereof. In one embodiment,the buffer solution is phosphate buffered saline (PBS). The pH andosmolarity of the buffer solution containing the dsRNA can be adjustedsuch that it is suitable for administering to a subject. Additionalbuffers are described above.

Alternatively, a dsRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.Additional liposomal formulations are described herein.

Subjects that would benefit from a reduction and/or inhibition ofAPOC3gene expression are those having elevated triglyceride levels,e.g., TG>150 mg/dL or those with severe hypertriglyceridemia, e.g.,TG>500 mg/dL. In one embodiment, a subject has an APOC3 gene variantwith a gain of function mutation. In other embodiments, the patient hasmixed HTG (Type V) decreased LPL activity and/or Familial HTG (IV)inactivating LPL mutations and/or familial combined increased ApoB-100levels. In another embodiments, the subject has uncontrolledhypertriglyceridemia with acute pancreatitis, or the subject is an HIVpatient on therapy, or the subject has a high fat diet (postprandialhypertriglyceridemia), or a metabolic syndrome, or compound treatment(retinoid therapy), or insulin resistance. Treatment of a subject thatwould benefit from a reduction and/or inhibition of APOC3 geneexpression includes therapeutic and prophylactic treatment.

The invention further provides methods for the use of a dsRNA or apharmaceutical composition thereof, e.g., for treating a subject thatwould benefit from reduction and/or inhibition of APOC3 expression,e.g., a subject having elevated triglyceride levels, in combination withother pharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating elevated triglyceridelevels. For example, in certain embodiments, a dsRNA targeting APOC3 isadministered in combination with, e.g., an agent useful in treatingelevated triglyceride levels. For example, additional therapeutics andtherapeutic methods suitable for treating a subject that would benefitfrom reduction in APOC3 expression, e.g., a subject having elevatedtriglyceride levels, include lifestyle and diet modification,prescription grade fish oil, fibrates, niacin, ApoC3 antisense, CETPinhibitors, bile acid sequestrants, nicotinic acid, HMG CoA reductaseinhibitors, Gemfibrozil, Fenofibrate, Cholesterol absorption inhibitors,neomycin, omega 3 fatty acids, and the like. The dsRNA and an additionaltherapeutic agent and/or treatment may be administered at the same timeand/or in the same combination, e.g., parenterally, or the additionaltherapeutic agent can be administered as part of a separate compositionor at separate times and/or by another method known in the art ordescribed herein.

In one embodiment, the method includes administering a compositionfeatured herein such that expression of the target APOC3 gene isdecreased for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, or 24 hours or28, 32, or 36 hours. In one embodiment, expression of the target APOC3gene is decreased for an extended duration, e.g., at least about two,three, four days or more, e.g., about one week, two weeks, three weeks,or four weeks or longer.

Administration of the dsRNA according to the methods of the inventionmay result in a reduction of the severity, signs, symptoms, and/ormarkers of such diseases or disorders in a patient with elevatedtriglyceride levels. By “reduction” in this context is meant astatistically significant decrease in such level. The reduction can be,for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, quality of life, dose of a medication required to sustain atreatment effect, level of a disease marker or any other measurableparameter appropriate for a given disease being treated or targeted forprevention. It is well within the ability of one skilled in the art tomonitor efficacy of treatment or prevention by measuring any one of suchparameters, or any combination of parameters. For example, efficacy oftreatment of elevated triglyceride levels may be assessed, for example,by periodic measurement of serum triglyceride levels. Comparisons of thelater readings with the initial readings provide a physician anindication of whether the treatment is effective. It is well within theability of one skilled in the art to monitor efficacy of treatment orprevention by measuring any one of such parameters, or any combinationof parameters. In connection with the administration of a dsRNAtargeting APOC3 or pharmaceutical composition thereof, “effectiveagainst” an elevated triglyceride levels indicates that administrationin a clinically appropriate manner results in a beneficial effect for atleast a statistically significant fraction of patients, such as aimprovement of symptoms, a cure, a reduction in disease, extension oflife, improvement in quality of life, or other effect generallyrecognized as positive by medical doctors familiar with elevatedtriglyceride levels and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given dsRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale, as butone example the Child-Pugh score (sometimes the Child-Turcotte-Pughscore). Any positive change resulting in e.g., lessening of severity ofdisease measured using the appropriate scale, represents adequatetreatment using a dsRNA or dsRNA formulation as described herein.

Subjects can be administered a therapeutic amount of dsRNA, such asabout 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, 0.5mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg,1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kg dsRNA, 2.9mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kg dsRNA, 3.3mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kg dsRNA, 3.7mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kg dsRNA, 4.1mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kg dsRNA, 4.5mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kg dsRNA, 4.9mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kg dsRNA, 5.3mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kg dsRNA, 5.7mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kg dsRNA, 6.1mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kg dsRNA, 6.5mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kg dsRNA, 6.9mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kg dsRNA, 7.3mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kg dsRNA, 7.7mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kg dsRNA, 8.1mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4 mg/kg dsRNA, 8.5mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kg dsRNA, 8.9mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kg dsRNA, 9.3mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kg dsRNA, 9.7mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 10 mg/kgdsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA, 30 mg/kg dsRNA,35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about 50 mg/kg dsRNA.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of dsRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg,about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In other embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and an N-acetylgalactosamine,subjects can be administered a therapeutic amount of dsRNA, such as adose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

For example, subjects can be administered a therapeutic amount of dsRNA,such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, orabout 50 mg/kg. Values and ranges intermediate to the recited values arealso intended to be part of this invention.

The dsRNA can be administered by intravenous infusion over a period oftime, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or about a 25 minute period. The administrationmay be repeated, for example, on a regular basis, such as biweekly(i.e., every two weeks) for one month, two months, three months, fourmonths or longer. After an initial treatment regimen, the treatments canbe administered on a less frequent basis. For example, afteradministration biweekly for three months, administration can be repeatedonce per month, for six months or a year or longer. Administration ofthe dsRNA can reduce APOC3 levels, e.g., in a cell, tissue, blood, urineor other compartment of the patient by at least about 5%, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, orat least about 99% or more.

Before administration of a full dose of the dsRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction. In anotherexample, the patient can be monitored for unwanted immunostimulatoryeffects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)levels.

Owing to the inhibitory effects on APOC3 expression, a compositionaccording to the invention or a pharmaceutical composition preparedthere from can enhance the quality of life.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the dsRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1 dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500A, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table B.

TABLE B Abbreviations. Abbreviation Nucleotide(s) Aadenosine-3′-phosphate C cytidine-3′-phosphate G guanosine-3′-phosphateU uridine-3′-phosphate N any nucleotide (G, A, C, or T) a2′-O-methyladenosine-3′-phosphate c 2′-O-methylcytidine-3′-phosphate g2′-O-methylguanosine-3′-phosphate u 2′-O-methyluridine-3′-phosphate T,dT 2′-deoxythymidine-3′-phosphate sT; sdT2′-deoxy-thymidine-5′phosphate-phosphorothioate

Example 2 APOC3 siRNA Design

Transcripts

siRNA design was carried out to identify siRNAs targeting all human andcynomolgus monkey (Macaca fascicularis; henceforth “cyno”) APOC3transcripts annotated in the NCBI Gene database(http://www.ncbi.nlm.nih.gov/gene/). Design used the followingtranscripts from NCBI: Human—NM_000040.1; cyno—X68359.1. All siRNAduplexes were designed that shared 100% identity with the listed humanand cyno transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The siRNAs were selected based on predicted specificity, predictedefficacy, and GC content.

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were then selected that lacked repeats longerthan 7 nucleotides. These 171 candidate siRNAs were used in acomprehensive search against the human transcriptome (defined as the setof NM_and XM_records within the human NCBI Refseq set)

A score was calculated based on the position and number of mismatchesbetween the siRNA and any potential ‘off-target’ transcript andcomparing the frequency of heptamers and octomers derived from 3distinct, seed (positions 2-9 from the 5′ end of the molecule)-derivedhexamers of each oligo. Both siRNAs strands were assigned to a categoryof specificity according to the calculated scores: a score above 3qualifies as highly specific, equal to 3 as specific and between 2.2 and2.8 as moderately specific. We sorted by the specificity of theantisense strand. We then selected duplexes whose antisense oligos hadless than 70% overall GC content, lacked GC at the first position, anddid not match the mouse APOC3 transcript NM 023114.3.

siRNA Sequence Selection

A total of 27 sense and 27 antisense derived siRNA oligos weresynthesized and formed into duplexes.

Example 3 APOC3 siRNA Synthesis

Synthesis of Modified and Unmodified ApoC3 Sequences

APOC3 tiled sequences were synthesized on MerMade 192 synthesizer ateither 1 or 0.2 umol scale.

Single strands and duplexes were made with either unmodified,2′-O-Methyl or 2′-fluoro chemical modifications. Synthesis conditionswere appropriately modified based on the nature of chemicalmodifications in the single strands.

Synthesis, Cleavage and Deprotection:

The synthesis of APOC3 sequences (unmodified, 2-O-Methyl or 2′-fluoro)used solid supported oligonucleotide synthesis using phosphoramiditechemistry.

The synthesis of the above sequences was performed at either 1 or 0.2 umscale in 96 well plates. Unmodified and modified (2-O-Methyl or2′-fluoro) amidite solutions were prepared at 0.1M concentration andethyl thio tetrazole (0.6M in Acetonitrile) was used as activator.

The synthesized sequences were cleaved and deprotected in 96 wellplates, using either aqueous ammonia or aqueous methylamine in the firststep and fluoride reagent in the second step. The crude sequences wereprecipitated using acetone:ethanol (80:20) mix and the pellet werere-suspended in 0.2M sodium acetate buffer to convert the crude singlestrands to their sodium salts. Samples from each sequence were analyzedby LC-MS to confirm the identity, UV for quantification and by IEXchromatography to determine purity.

Purification and Desalting:

APOC3 tiled sequences were precipitated and purified on AKTA Purifiersystem using Sephadex column. The process was run at ambienttemperature. Sample injection and collection was performed in 96 well(1.8 mL-deep well) plates. A single peak corresponding to the fulllength sequence was collected in the eluent. The desalted APOC3sequences were analyzed for concentration (by UV measurement at A260)and purity (by ion exchange HPLC). The complementary single strands werethen combined in a 1:1 stoichiometric ratio to form siRNA duplexes.

Tables 1 and 2 provide a first set of unmodified and modified sequences.

Example 4 APOC3 siRNA In Vitro Screening

Cell Culture and Transfections:

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in RPMI (ATCC) supplemented with 10% FBS,streptomycin, and glutamine (ATCC) before being released from the plateby trypsinization. Transfection was carried out by adding 14.8 μl ofOpti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well intoa 96-well plate and incubated at room temperature for 15 minutes. 80 μlof complete growth media without antibiotic containing ˜2×10⁴ Hep3Bcells were then added to the siRNA mixture. Cells were incubated foreither 24 or 120 hours prior to RNA purification. Single doseexperiments were performed at 10 nM and 0.1 nM final duplexconcentration and dose response experiments were done at 10, 1, 0.5,0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, 0.00001 nM finalduplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12).

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixingspeed was the same throughout the process). Ten microliters of magneticbeads and 80 μl Lysis/Binding Buffer mixture were added to a roundbottom plate and mixed for 1 minute. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After removing supernatant, the lysed cells were added to theremaining beads and mixed for 5 minutes. After removing supernatant,magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixedfor 1 minute. Beads were capture again and supernatant removed. Beadswere then washed with 150 μl Wash Buffer B, captured and supernatant wasremoved. Beads were next washed with 150 μl Elution Buffer, captured andsupernatant removed. Beads were allowed to dry for 2 minutes. Afterdrying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70°C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant wasremoved and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers,1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E), 0.5 μl ApoC3 TaqMan probe(Applied Biosystems cat #Hs00163644_m1) and 5 μl Lightcycler 480 probemaster mix (Roche Cat #04887301001) per well in a 384 well 50 plates(Roche cat #04887301001). Real time PCR was done in an ABI 7900HT RealTime PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Eachduplex was tested in two independent transfections and each transfectionwas assayed in duplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. IC50s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with AD-1955 or naïve cells over the same dose range,or to its own lowest dose.

Viability Screens

Cell viability was measured on days 3, 5 in HeLa and Hep3B cellsfollowing transfection with 100, 10, 1, 0.1, 0.01 and 0.0001 nM siRNA.Cells were plated at a density of 10,000 cells per well in 96 wellplates. Each siRNA was assayed in triplicate and the data averaged.siRNAs targeting PLK1 and AD-19200 were included as positive controlsfor loss of viability and AD-1955 as a negative control. PLK1 andAD-19200 result in a dose dependant loss of viability. To measureviability, 20 ul of CellTiter Blue (Promega) was added to each well ofthe 96 well plates after 3, 5, days and incubated at 37° C. for 2 hours.Plates were then read in a Spectrophotomoeter (Molecular Devices) at560Ex/590Em. Viability was expressed as the average value of light unitsfrom three replicate transfections+/−standard deviation. In some cases,relative viability was assessed by first averaging the three replicatetransfections and then normalizing to the values obtained from thelowest dose (0.001 nM).

The results are provided in Tables 3, 4, and 5.

Example 5 APOC3 In Vivo Testing in Mice

An siRNA targeting APOC3 was administered to mice, both wild type (5.0mg/kg) and a transgenic hyperlipidemic model SREBPtg/LDLR−/− KO mice(1.0 mg/kg). Mice were sacrificed two days after administration andhepatic target mRNA, serum triglycerides, and serum total cholesterollevels were determined. An MC3 containing LNp11 formulation was used.

The results for wild-type mice are shown in FIG. 1. Administration ofthe siRNA targeting APOC3 resulted in a knock down in mRNA levels, a 50%lowering of triglycerides, and a lowering of total cholesterol in wildtype mice. Administration of the siRNA targeting APOC3 resulted in a 80%lowering of triglycerides in the hyperlipidemic model mice, data notshown. The results demonstrate that APOC3 is a validated target forsiRNA based treatment of hypertriglyceridemia, including coronary heartdisease (CAD) and pancreatitis.

Example 6 Synthesis and Screening of Modified siRNA Targeting APOC3(Second Set)

Additional modified APOC3 siRNA were synthesized as described in Table 6and Table 7 using the methods described above. A UMdTdsdT modificationpattern is a dT-phosphorothioate-dT addition to each strand. A DECAFmodification pattern is as follows: Sense strand—2′O-methyls on allpyrimidines, dTsdT/dTdT overhang; Antisense strand—modify ‘U’ at any twosites in the dinucleotide motif UU/UA/UG in the seed region (positions2-9) plus 2′O-Methyls on last 3 nucleotides (positions 17-19) plus2′O-Methyls on every ‘U’ in positions 10-16; dTsdT/dTdT overhang. A FOMEmodification pattern is as follows: Sense strand—2′F (5′ first base)then alternating with 2′OMe, Antisense strand—2′OMe (5′first base), thenalternating with 2′F.

The siRNA described in Table 6 and Table 7 were assayed in Hep3b cellsas described above. The results are shown in Table 8.

Example 7 Synthesis and Screening of Modified siRNA Targeting APOC3(Third Set)

Additional modified APOC3 siRNA were synthesized as described in Table 9and Table 10 using the methods described above. The siRNA were assayedin Hep3b cells as described above. The results are shown in Table 11.

TABLE 1  ApoC3 siRNA (first set): unmodified sequences SEQ SEQPosition in ID ID Duplex name NM_000040.1 NO: Sense Sequence NO:Antisense Sequence AD-24548.1UM 264-282 2 ACUGGAGCACCGUUAAGGA 83UCCUUAACGGUGCUCCAGU AD-24549.1UM 417-435 3 GCCCCUGUAGGUUGCUUAA 84UUAAGCAACCUACAGGGGC AD-24550.1UM 418-436 4 CCCCUGUAGGUUGCUUAAA 85UUUAAGCAACCUACAGGGG AD-24551.1UM 47-65 5 AUGCAGCCCCGGGUACUCC 86GGAGUACCCGGGGCUGCAU AD-24552.1UM 412-430 6 GGGCUGCCCCUGUAGGUUG 87CAACCUACAGGGGCAGCCC AD-24553.1UM 267-285 7 GGAGCACCGUUAAGGACAA 88UUGUCCUUAACGGUGCUCC AD-24554.1UM 266-284 8 UGGAGCACCGUUAAGGACA 89UGUCCUUAACGGUGCUCCA AD-24555.1UM 423-441 9 GUAGGUUGCUUAAAAGGGA 90UCCCUUUUAAGCAACCUAC AD-24556.1UM 265-283 10 CUGGAGCACCGUUAAGGAC 91GUCCUUAACGGUGCUCCAG AD-24557.1UM 45-63 11 CCAUGCAGCCCCGGGUACU 92AGUACCCGGGGCUGCAUGG AD-24558.1UM 416-434 12 UGCCCCUGUAGGUUGCUUA 93UAAGCAACCUACAGGGGCA AD-24559.1UM 44-62 13 GCCAUGCAGCCCCGGGUAC 94GUACCCGGGGCUGCAUGGC AD-24560.1UM 263-281 14 UACUGGAGCACCGUUAAGG 95CCUUAACGGUGCUCCAGUA AD-24561.1UM 262-280 15 CUACUGGAGCACCGUUAAG 96CUUAACGGUGCUCCAGUAG AD-24562.1UM 261-279 16 ACUACUGGAGCACCGUUAA 97UUAACGGUGCUCCAGUAGU AD-24563.1UM 260-278 17 GACUACUGGAGCACCGUUA 98UAACGGUGCUCCAGUAGUC AD-24564.1UM 341-359 18 GCCUGAGACCUCAAUACCC 99GGGUAUUGAGGUCUCAGGC AD-24565.1UM 340-358 19 UGCCUGAGACCUCAAUACC 100GGUAUUGAGGUCUCAGGCA AD-24566.1UM 46-64 20 CAUGCAGCCCCGGGUACUC 101GAGUACCCGGGGCUGCAUG AD-24567.1UM 342-360 21 CCUGAGACCUCAAUACCCC 102GGGGUAUUGAGGUCUCAGG AD-24568.1UM 345-363 22 GAGACCUCAAUACCCCAAG 103CUUGGGGUAUUGAGGUCUC AD-24569.1UM 249-267 23 GUUCCCUGAAAGACUACUG 104CAGUAGUCUUUCAGGGAAC AD-24570.1UM 411-429 24 AGGGCUGCCCCUGUAGGUU 105AACCUACAGGGGCAGCCCU AD-24571.1UM 339-357 25 CUGCCUGAGACCUCAAUAC 106GUAUUGAGGUCUCAGGCAG AD-24572.1UM 351-369 26 UCAAUACCCCAAGUCCACC 107GGUGGACUUGGGGUAUUGA AD-24573.1UM 235-253 27 GACCGAUGGCUUCAGUUCC 108GGAACUGAAGCCAUCGGUC AD-24574.1UM 248-266 28 AGUUCCCUGAAAGACUACU 109AGUAGUCUUUCAGGGAACU AD-24575.1UM 415-433 29 CUGCCCCUGUAGGUUGCUU 110AAGCAACCUACAGGGGCAG AD-24576.1UM 234-252 30 UGACCGAUGGCUUCAGUUC 111GAACUGAAGCCAUCGGUCA AD-24577.1UM 168-186 31 AGACCGCCAAGGAUGCACU 112AGUGCAUCCUUGGCGGUCU AD-45078.1UM 232-250 32 GGUGACCGAUGGCUUCAGU 113ACUGAAGCCAUCGGUCACCTT AD-45084.1UM 237-255 33 CCGAUGGCUUCAGUUCCCU 114AGGGAACUGAAGCCAUCGGTT AD-45090.1UM 239-257 34 GAUGGCUUCAGUUCCCUGA 115UCAGGGAACUGAAGCCAUCTT AD-45096.1UM 240-258 35 AUGGCUUCAGUUCCCUGAA 116UUCAGGGAACUGAAGCCAUTT AD-45101.1UM 48-66 36 UGCAGCCCCGGGUACUCCU 117AGGAGUACCCGGGGCUGCATT AD-45102.1UM 241-259 37 UGGCUUCAGUUCCCUGAAA 118UUUCAGGGAACUGAAGCCATT AD-45107.1UM 49-67 38 GCAGCCCCGGGUACUCCUU 119AAGGAGUACCCGGGGCUGCTT AD-45108.1UM 243-261 39 GCUUCAGUUCCCUGAAAGA 120UCUUUCAGGGAACUGAAGCTT AD-45113.1UM 166-184 40 CAAGACCGCCAAGGAUGCA 121UGCAUCCUUGGCGGUCUUGTT AD-45114.1UM 251-269 41 UCCCUGAAAGACUACUGGA 122UCCAGUAGUCUUUCAGGGATT AD-45119.1UM 230-248 42 UGGGUGACCGAUGGCUUCA 123UGAAGCCAUCGGUCACCCATT AD-45120.1UM 254-272 43 CUGAAAGACUACUGGAGCA 124UGCUCCAGUAGUCUUUCAGTT AD-45121.1UM 259-277 44 AGACUACUGGAGCACCGUU 125AACGGUGCUCCAGUAGUCU AD-45122.1UM 410-428 45 CAGGGCUGCCCCUGUAGGU 126ACCUACAGGGGCAGCCCUG AD-45123.1UM 49-67 46 GCAGCCCCGGGUACUCCUU 127AAGGAGUACCCGGGGCUGC AD-45124.1UM 243-261 47 GCUUCAGUUCCCUGAAAGA 128UCUUUCAGGGAACUGAAGC AD-45125.1UM 343-361 48 CUGAGACCUCAAUACCCCA 129UGGGGUAUUGAGGUCUCAG AD-45126.1UM 430-448 49 GCUUAAAAGGGACAGUAUU 130AAUACUGUCCCUUUUAAGC AD-45127.1UM 269-287 50 AGCACCGUUAAGGACAAGU 131ACUUGUCCUUAACGGUGCU AD-45128.1UM 414-432 51 GCUGCCCCUGUAGGUUGCU 132AGCAACCUACAGGGGCAGC AD-45129.1UM 166-184 52 CAAGACCGCCAAGGAUGCA 133UGCAUCCUUGGCGGUCUUG AD-45130.1UM 251-269 53 UCCCUGAAAGACUACUGGA 134UCCAGUAGUCUUUCAGGGA AD-45131.1UM 344-362 54 UGAGACCUCAAUACCCCAA 135UUGGGGUAUUGAGGUCUCA AD-45132.1UM 514-532 55 CUGGACAAGAAGCUGCUAU 136AUAGCAGCUUCUUGUCCAG AD-45133.1UM 270-288 56 GCACCGUUAAGGACAAGUU 137AACUUGUCCUUAACGGUGC AD-45135.1UM 230-248 57 UGGGUGACCGAUGGCUUCA 138UGAAGCCAUCGGUCACCCA AD-45136.1UM 254-272 58 CUGAAAGACUACUGGAGCA 139UGCUCCAGUAGUCUUUCAG AD-45137.1UM 349-367 59 CCUCAAUACCCCAAGUCCA 140UGGACUUGGGGUAUUGAGG AD-45138.1UM 337-355 60 GGCUGCCUGAGACCUCAAU 141AUUGAGGUCUCAGGCAGCC AD-45139.1UM 425-443 61 AGGUUGCUUAAAAGGGACA 142UGUCCCUUUUAAGCAACCU AD-45140.1UM 232-250 62 GGUGACCGAUGGCUUCAGU 143ACUGAAGCCAUCGGUCACC AD-45141.1UM 259-277 63 AGACUACUGGAGCACCGUU 144AACGGUGCUCCAGUAGUCU AD-45143.1UM 338-356 64 GCUGCCUGAGACCUCAAUA 145UAUUGAGGUCUCAGGCAGC AD-45144.1UM 429-447 65 UGCUUAAAAGGGACAGUAU 146AUACUGUCCCUUUUAAGCA AD-45145.1UM 237-255 66 CCGAUGGCUUCAGUUCCCU 147AGGGAACUGAAGCCAUCGG AD-45146.1UM 269-287 67 AGCACCGUUAAGGACAAGU 148ACUUGUCCUUAACGGUGCU AD-45147.1UM 414-432 68 GCUGCCCCUGUAGGUUGCU 149AGCAACCUACAGGGGCAGC AD-45148.1UM 343-361 69 CUGAGACCUCAAUACCCCA 150UGGGGUAUUGAGGUCUCAG AD-45149.1UM 430-448 70 GCUUAAAAGGGACAGUAUU 151AAUACUGUCCCUUUUAAGC AD-45150.1UM 239-257 71 GAUGGCUUCAGUUCCCUGA 152UCAGGGAACUGAAGCCAUC AD-45151.1UM 270-288 72 GCACCGUUAAGGACAAGUU 153AACUUGUCCUUAACGGUGC AD-45152.1UM 419-437 73 CCCUGUAGGUUGCUUAAAA 154UUUUAAGCAACCUACAGGG AD-45153.1UM 344-362 74 UGAGACCUCAAUACCCCAA 155UUGGGGUAUUGAGGUCUCA AD-45154.1UM 514-532 75 CUGGACAAGAAGCUGCUAU 156AUAGCAGCUUCUUGUCCAG AD-45155.1UM 240-258 76 AUGGCUUCAGUUCCCUGAA 157UUCAGGGAACUGAAGCCAU AD-45157.1UM 425-443 77 AGGUUGCUUAAAAGGGACA 158UGUCCCUUUUAAGCAACCU AD-45158.1UM 349-367 78 CCUCAAUACCCCAAGUCCA 159UGGACUUGGGGUAUUGAGG AD-45159.1UM 48-66 79 UGCAGCCCCGGGUACUCCU 160AGGAGUACCCGGGGCUGCA AD-45160.1UM 241-259 80 UGGCUUCAGUUCCCUGAAA 161UUUCAGGGAACUGAAGCCA AD-45161.1UM 338-356 81 GCUGCCUGAGACCUCAAUA 162UAUUGAGGUCUCAGGCAGC AD-45162.1UM 429-447 82 UGCUUAAAAGGGACAGUAU 163AUACUGUCCCUUUUAAGCA

TABLE 2  ApoC3 modified siRNA (first set) sequences SEQ SEQ Duplex ID IDname NO: Sense Sequence NO: Antisense Sequence AD-24548.1 164AcuGGAGcAccGuuAAGGAdTsdT 245 UCCUuAACGGUGCUCcAGUdTsdT AD-24549.1 165GccccuGuAGGuuGcuuAAdTsdT 246 UuAAGcAACCuAcAGGGGCdTsdT AD-24550.1 166ccccuGuAGGuuGcuuAAAdTsdT 247 UUuAAGcAACCuAcAGGGGdTsdT AD-24551.1 167AuGcAGccccGGGuAcuccdTsdT 248 GGAGuACCCGGGGCUGcAUdTsdT AD-24552.1 168GGGcuGccccuGuAGGuuGdTsdT 249 cAACCuAcAGGGGcAGCCCdTsdT AD-24553.1 169GGAGcAccGuuAAGGAcAAdTsdT 250 UUGUCCUuAACGGUGCUCCdTsdT AD-24554.1 170uGGAGcAccGuuAAGGAcAdTsdT 251 UGUCCUuAACGGUGCUCcAdTsdT AD-24556.1 171cuGGAGcAccGuuAAGGAcdTsdT 252 GUCCUuAACGGUGCUCcAGdTsdT AD-24557.1 172ccAuGcAGccccGGGuAcudTsdT 253 AGuACCCGGGGCUGcAUGGdTsdT AD-24558.1 173uGccccuGuAGGuuGcuuAdTsdT 254 uAAGcAACCuAcAGGGGcAdTsdT AD-24559.1 174GccAuGcAGccccGGGuAcdTsdT 255 GuACCCGGGGCUGcAUGGCdTsdT AD-24560.1 175uAcuGGAGcAccGuuAAGGdTsdT 256 CCUuAACGGUGCUCcAGuAdTsdT AD-24561.1 176cuAcuGGAGcAccGuuAAGdTsdT 257 CUuAACGGUGCUCcAGuAGdTsdT AD-24563.1 177GAcuAcuGGAGcAccGuuAdTsdT 258 uAACGGUGCUCcAGuAGUCdTsdT AD-24564.1 178GccuGAGAccucAAuAcccdTsdT 259 GGGuAUUGAGGUCUcAGGCdTsdT AD-24565.1 179uGccuGAGAccucAAuAccdTsdT 260 GGuAUUGAGGUCUcAGGcAdTsdT AD-24566.1 180cAuGcAGccccGGGuAcucdTsdT 261 GAGuACCCGGGGCUGcAUGdTsdT AD-24567.1 181ccuGAGAccucAAuAccccdTsdT 262 GGGGuAUUGAGGUCUcAGGdTsdT AD-24568.1 182GAGAccucAAuAccccAAGdTsdT 263 CUUGGGGuAUUGAGGUCUCdTsdT AD-24569.1 183GuucccuGAAAGAcuAcuGdTsdT 264 cAGuAGUCUUUcAGGGAACdTsdT AD-24570.1 184AGGGcuGccccuGuAGGuudTsdT 265 AACCuAcAGGGGcAGCCCUdTsdT AD-24571.1 185cuGccuGAGAccucAAuAcdTsdT 266 GuAUUGAGGUCUcAGGcAGdTsdT AD-24572.1 186ucAAuAccccAAGuccAccdTsdT 267 GGUGGACUUGGGGuAUUGAdTsdT AD-24573.1 187GAccGAuGGcuucAGuuccdTsdT 268 GGAACUGAAGCcAUCGGUCdTsdT AD-24574.1 188AGuucccuGAAAGAcuAcudTsdT 269 AGuAGUCUUUcAGGGAACUdTsdT AD-24575.1 189cuGccccuGuAGGuuGcuudTsdT 270 AAGcAACCuAcAGGGGcAGdTsdT AD-24576.1 190uGAccGAuGGcuucAGuucdTsdT 271 GAACUGAAGCcAUCGGUcAdTsdT AD-24577.1 191AGAccGccAAGGAuGcAcudTsdT 272 AGUGcAUCCUUGGCGGUCUdTsdT AD-24555.1 192GuAGGuuGcuuAAAAGGGAdTsdT 273 UCCCUUUuAAGcAACCuACdTsdT AD-24562.1 193AcuAcuGGAGcAccGuuAAdTsdT 274 UuAACGGUGCUCcAGuAGUdTsdT AD-45078.1 194GGuGAccGAuGGcuucAGudTsdT 275 ACUGAAGCcAUCGGUcACCdTsdT AD-45084.1 195ccGAuGGcuucAGuucccudTsdT 276 AGGGAACUGAAGCcAUCGGdTsdT AD-45090.1 196GAuGGcuucAGuucccuGAdTsdT 277 UcAGGGAACUGAAGCcAUCdTsdT AD-45096.1 197AuGGcuucAGuucccuGAAdTsdT 278 UUcAGGGAACUGAAGCcAUdTsdT AD-45101.1 198uGcAGccccGGGuAcuccudTsdT 279 AGGAGuACCCGGGGCUGcAdTsdT AD-45102.1 199uGGcuucAGuucccuGAAAdTsdT 280 UUUcAGGGAACUGAAGCcAdTsdT AD-45107.1 200GcAGccccGGGuAcuccuudTsdT 281 AAGGAGuACCCGGGGCUGCdTsdT AD-45108.1 201GcuucAGuucccuGAAAGAdTsdT 282 UCUUUcAGGGAACUGAAGCdTsdT AD-45113.1 202cAAGAccGccAAGGAuGcAdTsdT 283 UGcAUCCUUGGCGGUCUUGdTsdT AD-45114.1 203ucccuGAAAGAcuAcuGGAdTsdT 284 UCcAGuAGUCUUUcAGGGAdTsdT AD-45119.1 204uGGGuGAccGAuGGcuucAdTsdT 285 UGAAGCcAUCGGUcACCcAdTsdT AD-45120.1 205cuGAAAGAcuAcuGGAGcAdTsdT 286 UGCUCcAGuAGUCUUUcAGdTsdT AD-45121.1 206AGAcuAcuGGAGcAccGuudTsdT 287 AACGGUGCUCcAGuAGUCUdTsdT AD-45122.1 207cAGGGcuGccccuGuAGGudTsdT 288 ACCuAcAGGGGcAGCCCUGdTsdT AD-45123.1 208GCfAGCfCfCfCfGGGUfACfUfCfCfUfUfdTsdT 289 AAGGAGUfACCCGGGGCUGCdTsdTAD-45124.1 209 GCfUfUfCfAGUfUfCfCfCfUfGAAAGAdTsdT 290UCUUUCfAGGGAACUGAAGCdTsdT AD-45125.1 210CfUfGAGACfCfUfCfAAUfACfCfCfCfAdTsdT 291 UGGGGUfAUUGAGGUCUCfAGdTsdTAD-45126.1 211 GCfUfUfAAAAGGGACfAGUfAUfUfdTsdT 292AAUfACUGUCCCUUUUfAAGCdTsdT AD-45127.1 212 AGcAccGuuAAGGAcAAGudTsdT 293ACUUGUCCUuAACGGUGCUdTsdT AD-45128.1 213 GcuGccccuGuAGGuuGcudTsdT 294AGcAACCuAcAGGGGcAGCdTsdT AD-45129.1 214 CfAAGACfCfGCfCfAAGGAUfGCfAdTsdT295 UGCfAUCCUUGGCGGUCUUGdTsdT AD-45130.1 215UfCfCfCfUfGAAAGACfUfACfUfGGAdTsdT 296 UCCfAGUfAGUCUUUCfAGGGAdTsdTAD-45131.1 216 UfGAGACfCfUfCfAAUfACfCfCfCfAAdTsdT 297UUGGGGUfAUUGAGGUCUCfAdTsdT AD-45132.1 217CfUfGGACfAAGAAGCfUfGCfUfAUfdTsdT 298 AUfAGCfAGCUUCUUGUCCfAGdTsdTAD-45133.1 218 GcAccGuuAAGGAcAAGuudTsdT 299 AACUUGUCCUuAACGGUGCdTsdTAD-45135.1 219 UfGGGUfGACfCfGAUfGGCfUfUfCfAdTsdT 300UGAAGCCfAUCGGUCfACCCfAdTsdT AD-45136.1 220CfUfGAAAGACfUfACfUfGGAGCfAdTsdT 301 UGCUCCfAGUfAGUCUUUCfAGdTsdTAD-45137.1 221 CfCfUfCfAAUfACfCfCfCfAAGUfCfCfAdTsdT 302UGGACUUGGGGUfAUUGAGGdTsdT AD-45138.1 222 GGcuGccuGAGAccucAAudTsdT 303AUUGAGGUCUcAGGcAGCCdTsdT AD-45139.1 223 AGGuuGcuuAAAAGGGAcAdTsdT 304UGUCCCUUUuAAGcAACCUdTsdT AD-45140.1 224GGUfGACfCfGAUfGGCfUfUfCfAGUfdTsdT 305 ACUGAAGCCfAUCGGUCfACCdTsdTAD-45141.1 225 AGACfUfACfUfGGAGCfACfCfGUfUfdTsdT 306AACGGUGCUCCfAGUfAGUCUdTsdT AD-45143.1 226 GcuGccuGAGAccucAAuAdTsdT 307uAUUGAGGUCUcAGGcAGCdTsdT AD-45144.1 227 uGcuuAAAAGGGAcAGuAudTsdT 308AuACUGUCCCUUUuAAGcAdTsdT AD-45145.1 228CfCfGAUfGGCfUfUfCfAGUfUfCfCfCfUfdTsdT 309 AGGGAACUGAAGCCfAUCGGdTsdTAD-45146.1 229 AGCfACfCfGUfUfAAGGACfAAGUfdTsdT 310ACUUGUCCUUfAACGGUGCUdTsdT AD-45147.1 230GCfUfGCfCfCfCfUfGUfAGGUfUfGCfUfdTsdT 311 AGCfAACCUfACfAGGGGCfAGCdTsdTAD-45148.1 231 cuGAGAccucAAuAccccAdTsdT 312 UGGGGuAUUGAGGUCUcAGdTsdTAD-45149.1 232 GcuuAAAAGGGAcAGuAuudTsdT 313 AAuACUGUCCCUUUuAAGCdTsdTAD-45150.1 233 GAUfGGCfUfUfCfAGUfUfCfCfCfUfGAdTsdT 314UCfAGGGAACUGAAGCCfAUCdTsdT AD-45151.1 234GCfACfCfGUfUfAAGGACfAAGUfUfdTsdT 315 AACUUGUCCUUfAACGGUGCdTsdTAD-45152.1 235 CfCfCfUfGUfAGGUfUfGCfUfUfAAAAdTsdT 316UUUUfAAGCfAACCUfACfAGGGdTsdT AD-45153.1 236 uGAGAccucAAuAccccAAdTsdT 317UUGGGGuAUUGAGGUCUcAdTsdT AD-45154.1 237 cuGGAcAAGAAGcuGcuAudTsdT 318AuAGcAGCUUCUUGUCcAGdTsdT AD-45155.1 238AUfGGCfUfUfCfAGUfUfCfCfCfUfGAAdTsdT 319 UUCfAGGGAACUGAAGCCfAUdTsdTAD-45157.1 239 AGGUfUfGCfUfUfAAAAGGGACfAdTsdT 320UGUCCCUUUUfAAGCfAACCUdTsdT AD-45158.1 240 ccucAAuAccccAAGuccAdTsdT 321UGGACUUGGGGuAUUGAGGdTsdT AD-45159.1 241UfGCfAGCfCfCfCfGGGUfACfUfCfCfUfdTsdT 322 AGGAGUfACCCGGGGCUGCfAdTsdTAD-45160.1 242 UfGGCfUfUfCfAGUfUfCfCfCfUfGAAAdTsdT 323UUUCfAGGGAACUGAAGCCfAdTsdT AD-45161.1 243GCfUfGCfCfUfGAGACfCfUfCfAAUfAdTsdT 324 UfAUUGAGGUCUCfAGGCfAGCdTsdTAD-45162.1 244 UfGCfUfUfAAAAGGGACfAGUfAUfdTsdT 325AUfACUGUCCCUUUUfAAGCfAdTsdT Lowercase nucleotides (g, a, u, c) are2′-O-methyl nucleotides; Nf (e.g., Gf, Af, Uf, CO is a 2′-fluoronucleotide; s is a phosphothiorate linkage.

TABLE 3 ApoC3 modified siRNA (first set) single dose screen Duplex ID 10nM 0.1 nM AD-24548.1 0.06 0.38 AD-24549.1 0.17 0.39 AD-24550.1 0.38 0.67AD-24551.1 1.08 1.02 AD-24552.1 0.98 0.97 AD-24553.1 0.51 0.63AD-24554.1 0.63 0.78 AD-24555.1 0.06 0.29 AD-24556.1 0.17 0.72AD-24557.1 0.81 0.93 AD-24558.1 0.90 0.75 AD-24559.1 0.88 0.94AD-24560.1 0.75 0.85 AD-24561.1 0.40 0.77 AD-24562.1 0.07 0.39AD-24563.1 0.55 0.91 AD-24564.1 0.70 1.00 AD-24565.1 0.67 1.00AD-24566.1 0.97 1.01 AD-24567.1 0.89 0.92 AD-24568.1 0.95 0.85AD-24569.1 0.68 0.88 AD-24570.1 0.74 0.77 AD-24571.1 0.22 0.60AD-24572.1 0.92 0.91 AD-24573.1 0.65 0.76 AD-24574.1 0.70 0.80AD-24575.1 0.63 0.94 AD-24576.1 0.05 0.31 AD-24577.1 0.90 0.98AD-45078.1 0.38 0.78 AD-45084.1 0.60 0.92 AD-45090.1 0.97 0.86AD-45096.1 0.47 0.82 AD-45101.1 1.01 1.30 AD-45102.1 0.05 0.22AD-45107.1 0.87 1.06 AD-45108.1 0.02 0.12 AD-45113.1 0.97 1.04AD-45114.1 0.37 0.77 AD-45119.1 0.91 0.87 AD-45120.1 0.03 0.08AD-45121.1 0.93 0.94 AD-45122.1 0.92 0.97 AD-45123.1 0.15 0.41AD-45124.1 0.03 0.07 AD-45125.1 0.16 0.55 AD-45126.1 0.03 0.08AD-45127.1 0.58 0.75 AD-45128.1 0.97 0.96 AD-45129.1 0.20 0.47AD-45130.1 0.05 0.12 AD-45131.1 0.24 0.64 AD-45132.1 0.30 0.52AD-45133.1 0.03 0.10 AD-45135.1 0.02 0.08 AD-45136.1 0.04 0.10AD-45137.1 0.02 0.19 AD-45138.1 0.86 1.04 AD-45139.1 1.19 1.13AD-45140.1 0.07 0.33 AD-45141.1 0.03 0.07 AD-45143.1 0.73 0.94AD-45144.1 0.45 0.95 AD-45145.1 0.04 0.13 AD-45146.1 0.06 0.21AD-45147.1 0.20 0.49 AD-45148.1 0.80 0.94 AD-45149.1 0.03 0.07AD-45150.1 0.09 0.28 AD-45151.1 0.03 0.05 AD-45152.1 0.04 0.13AD-45153.1 0.92 1.02 AD-45154.1 0.14 0.29 AD-45155.1 0.60 0.68AD-45157.1 0.13 0.29 AD-45158.1 0.37 0.78 AD-45159.1 0.12 0.53AD-45160.1 0.03 0.11 AD-45161.1 0.59 0.54 AD-45162.1 0.02 0.06

TABLE 4 ApoC3 modified siRNA (first set) IC50 data DuplexName IC50 24 hr(nM) IC50 120 hr (nM) AD-24555 0.038 0.091 AD-24562 0.025 0.106 AD-245760.037 0.059 AD-45102.1 0.012 0.022 AD-45108.1 0.014 0.246 AD-45120.10.011 0.02 AD-45124.1 0.013 0.264 AD-45126.1 0.025 0.098 AD-45129.10.023 0.046 AD-45133.1 0.014 0.015 AD-45135.1 0.008 0.064 AD-45136.10.008 0.053 AD-45137.1 0.010 0.077 AD-45141.1 0.007 0.063 AD-45145.10.013 0.113 AD-45146.1 0.031 0.316 AD-45149.1 0.011 0.091 AD-45151.10.006 0.009 AD-45152.1 0.011 0.051 AD-45160.1 0.019 0.162 AD-45162.10.008 0.013

TABLE 5 ApoC3 modified siRNA (first set) viability Viability data areexpressed as fraction viable relative to cells treated with the lowestdose of siRNA (0.0001 nM). 1 = 100% viable, 0 = 100% lethality Fractionviable normalized to low dose (0.0001 nM) Conc. (nM) 10 nM 1 nM 0.1 nM0.01 nM 0.0001 nM HeLa day 3 AD-45102.1 0.57 0.72 0.96 1.06 1.00AD-45108.1 0.58 0.87 0.99 0.97 1.00 AD-45120.1 0.16 0.33 0.75 0.97 1.00AD-45124.1 0.69 0.84 0.96 0.94 1.00 AD-45126.1 0.47 0.46 0.65 0.95 1.00AD-45130.1 0.64 0.72 0.93 1.00 1.00 AD-45133.1 0.22 0.51 0.94 0.94 1.00AD-45151.1 0.43 0.63 1.12 1.06 1.00 AD-45152.1 0.70 0.96 1.02 1.06 1.00AD-45160.1 0.44 0.68 0.83 0.99 1.00 AD-45162.1 0.62 0.86 1.01 1.01 1.00AD-24555 0.67 0.91 1.00 0.96 1.00 AD-24562 0.59 0.74 0.82 0.92 1.00AD-24576 0.39 0.71 1.01 0.91 1.00 AD-45135.1 0.18 0.43 0.94 1.02 1.00AD-45136.1 0.33 0.48 0.86 1.00 1.00 AD-45137.1 0.65 0.89 0.96 0.91 1.00AD-45141.1 0.51 0.53 0.88 0.98 1.00 AD-45145.1 0.33 0.58 0.95 0.92 1.00AD-45146.1 0.39 0.47 0.87 0.93 1.00 AD-45149.1 0.57 0.64 0.96 0.96 1.00AD-1955 0.62 0.84 0.93 0.99 1.00 PLK 0.02 0.05 0.12 0.62 1.00 AD-192000.15 0.34 0.81 0.93 1.00 HeLa Day 5 AD-45102.1 0.55 0.79 0.89 1.00 1.00AD-45108.1 0.77 0.95 0.99 1.01 1.00 AD-45120.1 0.06 0.28 0.90 1.00 1.00AD-45124.1 1.12 1.13 1.02 1.08 1.00 AD-45126.1 0.84 0.87 0.98 1.04 1.00AD-45130.1 0.50 0.81 1.04 1.11 1.00 AD-45133.1 0.01 0.11 0.76 0.94 1.00AD-45151.1 0.17 0.41 0.63 1.00 1.00 AD-45152.1 0.82 0.97 0.84 1.01 1.00AD-45160.1 0.47 0.83 0.94 1.03 1.00 AD-45162.1 0.79 0.94 0.83 1.00 1.00AD-24555 0.92 1.04 0.99 0.99 1.00 AD-24562 0.71 0.98 1.05 1.03 1.00AD-24576 0.10 0.59 0.80 1.00 1.00 AD-45135.1 0.04 0.66 1.02 1.02 1.00AD-45136.1 0.23 0.67 1.06 0.96 1.00 AD-45137.1 0.73 0.93 1.02 0.98 1.00AD-45141.1 0.30 0.51 0.91 0.97 1.00 AD-45145.1 0.27 0.76 1.01 1.01 1.00AD-45146.1 0.29 0.59 0.98 1.02 1.00 AD-45149.1 0.71 0.84 1.01 0.99 1.00AD-1955 0.67 0.89 0.92 0.95 1.00 PLK −0.03 0.02 0.06 0.47 0.88 AD-192000.05 0.49 1.01 1.03 1.00 Hep3B Day 3 AD-45102.1 0.84 1.09 1.02 1.06 1.00AD-45108.1 0.88 1.02 0.99 0.96 1.00 AD-45120.1 0.69 0.99 0.99 0.94 1.00AD-45124.1 0.86 1.09 0.95 0.92 1.00 AD-45126.1 0.73 0.95 0.99 0.97 1.00AD-45130.1 0.81 1.00 1.04 1.00 1.00 AD-45133.1 0.64 0.98 1.05 1.02 1.00AD-45151.1 0.53 0.70 0.91 0.86 1.00 AD-45152.1 0.86 0.93 0.98 1.02 1.00AD-45160.1 1.03 1.11 1.00 0.95 1.00 AD-45162.1 0.91 0.95 1.02 0.96 1.00AD-24555 0.83 0.82 0.93 0.81 1.00 AD-24562 1.14 1.26 1.15 1.03 1.00AD-24576 0.84 1.06 1.11 1.00 1.00 AD-45135.1 0.99 1.18 1.17 1.18 1.00AD-45136.1 0.83 0.98 1.05 1.12 1.00 AD-45137.1 0.93 1.12 1.04 1.03 1.00AD-45141.1 0.71 0.89 0.93 1.12 1.00 AD-45145.1 0.87 1.07 1.03 1.05 1.00AD-45146.1 0.85 1.01 1.07 1.09 1.00 AD-45149.1 0.98 1.20 1.10 1.04 1.00AD-1955 0.62 0.92 0.95 0.93 1.00 PLK 0.21 0.32 0.47 0.82 1.00 AD-192000.25 0.63 1.03 1.01 1.00 Hep3B Day 5 AD-45102.1 0.73 0.96 1.03 0.94 1.00AD-45108.1 1.01 0.83 0.96 0.96 1.00 AD-45120.1 0.30 0.47 0.81 1.00 1.00AD-45124.1 1.33 1.24 0.89 1.04 1.00 AD-45126.1 1.08 1.05 1.00 0.92 1.00AD-45130.1 0.86 0.92 1.09 0.93 1.00 AD-45133.1 0.47 0.58 0.93 0.95 1.00AD-45151.1 0.29 0.57 0.93 0.91 1.00 AD-45152.1 1.00 0.94 0.93 0.96 1.00AD-45160.1 1.46 1.25 1.20 0.90 1.00 AD-45162.1 0.83 0.84 0.89 0.85 1.00AD-24555 1.13 1.00 0.99 0.83 1.00 AD-24562 1.16 1.13 1.03 0.97 1.00AD-24576 0.68 0.92 1.04 0.90 1.00 AD-45135.1 0.81 1.23 1.35 1.19 1.00AD-45136.1 0.37 0.74 0.92 1.00 1.00 AD-45137.1 0.74 0.90 0.99 0.96 1.00AD-45141.1 0.32 0.43 0.61 0.96 1.00 AD-45145.1 0.52 0.74 0.96 1.00 1.00AD-45146.1 0.60 0.57 0.86 1.02 1.00 AD-45149.1 0.83 0.94 1.01 0.97 1.00AD-1955 0.63 0.74 0.93 0.85 1.00 PLK 0.03 0.12 0.29 0.86 1.00 AD-19200−0.04 0.41 0.84 0.95 1.00

TABLE 6 ApoC3 siRNA (second set) unmodified sequences and duplex names of modified siRNASEQ SEQ Modified Modifi- Position Unmodified ID ID Unmodified duplexcation in duplex name NO Unmodified sense NO antisense name TypeNM_000040.1 AD-45101.1UM 326 UGCAGCCCCGGGUACUCCU 353 AGGAGUACCCGGGGCUGCAAD-46822.1 end 48-66 AD-47334.1 FOME 48-66 AD-47361.1 DECAF 48-66AD-45107.1UM 327 GCAGCCCCGGGUACUCCUU 354 AAGGAGUACCCGGGGCUGC AD-46825.1UMdTsdT 49-67 AD-47338.1 FOME 49-67 AD-47365.1 DECAF 49-67 AD-45113.1UM328 CAAGACCGCCAAGGAUGCA 355 UGCAUCCUUGGCGGUCUUG  AD-46828.1 UMdTsdT166-184 AD-47342.1 FOME 166-184 AD-47369.1 DECAF 166-184 AD-45119.1UM329 UGGGUGACCGAUGGCUUCA 356 UGAAGCCAUCGGUCACCCA AD-46831.1  UMdTsdT230-248 AD-47346.1 FOME 230-248 AD-47373.1 DECAF 230-248 AD-45078.1UM330 GGUGACCGAUGGCUUCAGU 357 ACUGAAGCCAUCGGUCACC AD-46811.1  UMdTsdT232-250 AD-47349.1 FOME 232-250 AD-47376.1 DECAF 232-250 AD-45084.1UM331 CCGAUGGCUUCAGUUCCCU 358 AGGGAACUGAAGCCAUCGG  AD-46815.1 UMdTsdT237-255 AD-47352.1 FOME 237-255 AD-47379.1 DECAF 237-255 AD-45090.1UM332 GAUGGCUUCAGUUCCCUGA 359 UCAGGGAACUGAAGCCAUC AD-46818.1  UMdTsdT239-257 AD-47355.1 FOME 239-257 AD-47382.1 DECAF 239-257 AD-45096.1UM333 AUGGCUUCAGUUCCCUGAA 360 UUCAGGGAACUGAAGCCAU AD-46820.1  UMdTsdT240-258 AD-47358.1 FOME 240-258 AD-47385.1 DECAF 240-258 AD-45102.1UM334 UGGCUUCAGUUCCCUGAAA 361 UUUCAGGGAACUGAAGCCA AD-46823.1  UMdTsdT241-259 AD-47335.1 FOME 241-259 AD-47362.1 DECAF 241-259 AD-45108.1UM335 GCUUCAGUUCCCUGAAAGA 362 UCUUUCAGGGAACUGAAGC AD-46826.1  UMdTsdT243-261 AD-47339.1 FOME 243-261 AD-47366.1 DECAF 243-261 AD-45120.1UM336 CUGAAAGACUACUGGAGCA 363 UGCUCCAGUAGUCUUUCAG AD-46829.1 UMdTsdT254-272 AD-47347.1 FOME 254-272 AD-47374.1 DECAF 254-272 AD-45127.1UM337 AGCACCGUUAAGGACAAGU 364 ACUUGUCCUUAACGGUGCU AD-46832.1 UMdTsdT269-287 AD-47353.1 FOME 269-287 AD-47380.1 DECAF 269-287 AD-45133.1UM338 GCACCGUUAAGGACAAGUU 365 AACUUGUCCUUAACGGUGC AD-46812.1 UMdTsdT270-288 AD-47356.1 FOME 270-288 AD-47383.1 DECAF 270-288 AD-45143.1UM339 GCUGCCUGAGACCUCAAUA 366 UAUUGAGGUCUCAGGCAGC AD-46816.1 UMdTsdT338-356 AD-47336.1 FOME 338-356 AD-47363.1 DECAF 338-356 AD-45148.1UM340 CUGAGACCUCAAUACCCCA 367 UGGGGUAUUGAGGUCUCAG AD-46819.1 UMdTsdT343-361 AD-47340.1 FOME 343-361 AD-47367.1 DECAF 343-361 AD-45153.1UM341 UGAGACCUCAAUACCCCAA 368 UUGGGGUAUUGAGGUCUCA AD-46821.1 UMdTsdT344-362 AD-47344.1 FOME 344-362 AD-47371.1 DECAF 344-362 AD-45158.1UM342 CCUCAAUACCCCAAGUCCA 369 UGGACUUGGGGUAUUGAGG AD-46824.1 UMdTsdT349-367 AD-47348.1 FOME 349-367 AD-47375.1 DECAF 349-367 AD-45128.1UM343 GCUGCCCCUGUAGGUUGCU 370 AGCAACCUACAGGGGCAGC AD-46827.1 UMdTsdT414-432 AD-47354.1 FOME 414-432 AD-45139.1UM 344 AGGUUGCUUAAAAGGGACA 371UGUCCCUUUUAAGCAACCU AD-46830.1 UMdTsdT 425-443 AD-47360.1 FOME 425-443AD-47387.1 DECAF 425-443 AD-45144.1UM 345 UGCUUAAAAGGGACAGUAU 372AUACUGUCCCUUUUAAGCA AD-46833.1 UMdTsdT 429-447 AD-47337.1 FOME 429-447AD-47364.1 DECAF 429-447 AD-45149.1UM 346 GCUUAAAAGGGACAGUAUU 373AAUACUGUCCCUUUUAAGC AD-46813.1 UMdTsdT 430-448 AD-47341.1 FOME 430-448AD-47368.1 DECAF 430-448 AD-45154.1UM 347 CUGGACAAGAAGCUGCUAU 374AUAGCAGCUUCUUGUCCAG AD-46817.1 UMdTsdT 514-532 AD-47345.1 FOME 514-532AD-47372.1 DECAF 514-532 AD-45114.1UM 348 UCCCUGAAAGACUACUGGA 375UCCAGUAGUCUUUCAGGGA AD-47343.1 FOME 251-269 AD-45141.1UM 349AGACUACUGGAGCACCGUU 376 AACGGUGCUCCAGUAGUCU AD-47350.1 FOME 259-277AD-45138.1UM 350 GGCUGCCUGAGACCUCAAU 377 AUUGAGGUCUCAGGCAGCC AD-47359.1FOME 337-355 AD-47386.1 DECAF 337-355 AD-45122.1UM 351CAGGGCUGCCCCUGUAGGU 378 ACCUACAGGGGCAGCCCUG AD-47351.1 FOME 410-428AD-47378.1 DECAF 410-428 AD-45152.1UM 352 CCCUGUAGGUUGCUUAAAA 379UUUUAAGCAACCUACAGGG AD-47357.1 FOME 419-437 AD-47357.1 FOME 419-437

TABLE 7  ApoC3 modified siRNA (second set) sequences SEQ SEQ Modified IDID duplex name NO: Sense strand sequence 5′ to 3′ NO:Antisense strand sequence 5′ to 3′ AD-46822.1 380UGCAGCCCCGGGUACUCCUdTsdT 453 AGGAGUACCCGGGGCUGCAdTsdT AD-47334.1 381UfgCfaGfcCfcCfgGfgUfaCfuCfcUfdTsdT 454 aGfgAfgUfaCfcCfgGfgGfcUfgCfadTsdTAD-47361.1 382 uGcAGccccGGGuAcuccudTsdT 455 AGGAGuACCCGGGGCugcadTsdTAD-46825.1 383 GCAGCCCCGGGUACUCCUUdTsdT 456 AAGGAGUACCCGGGGCUGCdTsdTAD-47338.1 384 GfcAfgCfcCfcGfgGfuAfcUfcCfuUfdTsdT 457aAfgGfaGfuAfcCfcGfgGfgCfuGfcdTsdT AD-47365.1 385GcAGccccGGGuAcuccuudTsdT 458 AAGGAGuACCCGGGGCugcdTsdT AD-46828.1 386CAAGACCGCCAAGGAUGCAdTsdT 459 UGCAUCCUUGGCGGUCUUGdTsdT AD-47342.1 387CfaAfgAfcCfgCfcAfaGfgAfuGfcAfdTsdT 460 uGfcAfuCfcUfuGfgCfgGfuCfuUfgdTsdTAD-47369.1 388 cAAGAccGccAAGGAuGcAdTsdT 461 UGCAUCCuUGGCGGuCuugdTsdTAD-46831.1 389 UGGGUGACCGAUGGCUUCAdTsdT 462 UGAAGCCAUCGGUCACCCAdTsdTAD-47346.1 390 UfgGfgUfgAfcCfgAfuGfgCfuUfcAfdTsdT 463uGfaAfgCfcAfuCfgGfuCfaCfcCfadTsdT AD-47373.1 391uGGGuGAccGAuGGcuucAdTsdT 464 UGAAGCCAUCGGuCACccadTsdT AD-46811.1 392GGUGACCGAUGGCUUCAGUdTsdT 465 ACUGAAGCCAUCGGUCACCdTsdT AD-47349.1 393GfgUfgAfcCfgAfuGfgCfuUfcAfgUfdTsdT 466 aCfuGfaAfgCfcAfuCfgGfuCfaCfcdTsdTAD-47376.1 394 GGuGAccGAuGGcuucAGudTsdT 467 ACuGAAGCCAuCGGuCaccdTsdTAD-46815.1 395 CCGAUGGCUUCAGUUCCCUdTsdT 468 AGGGAACUGAAGCCAUCGGdTsdTAD-47352.1 396 CfcGfaUfgGfcUfuCfaGfuUfcCfcUfdTsdT 469aGfgGfaAfcUfgAfaGfcCfaUfcGfgdTsdT AD-47379.1 397ccGAuGGcuucAGuucccudTsdT 470 AGGGAACuGAAGCCAucggdTsdT AD-46818.1 398GAUGGCUUCAGUUCCCUGAdTsdT 471 UCAGGGAACUGAAGCCAUCdTsdT AD-47355.1 399GfaUfgGfcUfuCfaGfuUfcCfcUfgAfdTsdT 472 uCfaGfgGfaAfcUfgAfaGfcCfaUfcdTsdTAD-47382.1 400 GAuGGcuucAGuucccuGAdTsdT 473 UCAGGGAACuGAAGCCaucdTsdTAD-46820.1 401 AUGGCUUCAGUUCCCUGAAdTsdT 474 UUCAGGGAACUGAAGCCAUdTsdTAD-47358.1 402 AfuGfgCfuUfcAfgUfuCfcCfuGfaAfdTsdT 475uUfcAfgGfgAfaCfuGfaAfgCfcAfudTsdT AD-47385.1 403AuGGcuucAGuucccuGAAdTsdT 476 UUCAGGGAACuGAAGCcaudTsdT AD-46823.1 404UGGCUUCAGUUCCCUGAAAdTsdT 477 UUUCAGGGAACUGAAGCCAdTsdT AD-47335.1 405UfgGfcUfuCfaGfuUfcCfcUfgAfaAfdTsdT 478 uUfuCfaGfgGfaAfcUfgAfaGfcCfadTsdTAD-47362.1 406 uGGcuucAGuucccuGAAAdTsdT 479 UuUCAGGGAACuGAAGccadTsdTAD-46826.1 407 GCUUCAGUUCCCUGAAAGAdTsdT 480 UCUUUCAGGGAACUGAAGCdTsdTAD-47339.1 408 GfcUfuCfaGfuUfcCfcUfgAfaAfgAfdTsdT 481uCfuUfuCfaGfgGfaAfcUfgAfaGfcdTsdT AD-47366.1 409GcuucAGuucccuGAAAGAdTsdT 482 UCuUUCAGGGAACuGAagcdTsdT AD-46829.1 410CUGAAAGACUACUGGAGCAdTsdT 483 UGCUCCAGUAGUCUUUCAGdTsdT AD-47347.1 411CfuGfaAfaGfaCfuAfcUfgGfaGfcAfdTsdT 484 uGfcCfcCfaGfuAfgUfcUfuUfcAfgdTsdTAD-47374.1 412 cuGAAAGAcuAcuGGAGcAdTsdT 485 UGCUCCAGuAGuCuuucagdTsdTAD-46832.1 413 AGCACCGUUAAGGACAAGUdTsdT 486 ACUUGUCCUUAACGGUGCUdTsdTAD-47353.1 414 AfgCfaCfcGfuUfaAfgGfaCfaAfgUfdTsdT 487aCfuUfgUfcCfuUfaAfcGfgUfgCfudTsdT AD-47380.1 415AGcAccGuuAAGGAcAAGudTsdT 488 ACuUGUCCuUAACGGugcudTsdT AD-46812.1 416GCACCGUUAAGGACAAGUUdTsdT 489 AACUUGUCCUUAACGGUGCdTsdT AD-47356.1 417GfcAfcCfgUfuAfaGfgAfcAfaGfuUfdTsdT 490 aAfcUfuGfuCfcUfuAfaCfgGfuGfcdTsdTAD-47383.1 418 GcAccGuuAAGGAcAAGuudTsdT 491 AACuUGUCCuuAACGGugcdTsdTAD-46816.1 419 GCUGCCUGAGACCUCAAUAdTsdT 492 UAUUGAGGUCUCAGGCAGCdTsdTAD-47336.1 420 GfcUfgCfcUfgAfgAfcCfuCfaAfuAfdTsdT 493uAfuUfgAfgGfuCfuCfaGfgCfaGfcdTsdT AD-47363.1 421GcuGccuGAGAccucAAuAdTsdT 494 UAuUGAGGUCuCAGGCagcdTsdT AD-46819.1 422CUGAGACCUCAAUACCCCAdTsdT 495 UGGGGUAUUGAGGUCUCAGdTsdT AD-47340.1 423CfuGfaGfaCfcCfcAfaUfaCfcCfcAfdTsdT 496 uGfgGfgUfaUfuGfaGfgUfcCfcAfgdTsdTAD-47367.1 424 cuGAGAccucAAuAccccAdTsdT 497 UGGGGuAuUGAGGuCucagdTsdTAD-46821.1 425 UGAGACCUCAAUACCCCAAdTsdT 498 UUGGGGUAUUGAGGUCUCAdTsdTAD-47344.1 426 UfgAfgAfcCfuCfaAfuAfcCfcCfaAfdTsdT 499uUfgGfgGfuAfuUfgAfgGfuCfuCfadTsdT AD-47371.1 427uGAGAccucAAuAccccAAdTsdT 500 UuGGGGuAuUGAGGuCucadTsdT AD-46824.1 428CCUCAAUACCCCAAGUCCAdTsdT 501 UGGACUUGGGGUAUUGAGGdTsdT AD-47348.1 429CfcCfcAfaUfaCfcCfcAfaGfuCfcAfdTsdT 502 uGfgAfcUfuGfgGfgUfaUfuGfaGfgdTsdTAD-47375.1 430 ccucAAuAccccAAGuccAdTsdT 503 UGGACuUGGGGuAuuGaggdTsdTAD-46827.1 431 GCUGCCCCUGUAGGUUGCUdTsdT 504 AGCAACCUACAGGGGCAGCdTsdTAD-47354.1 432 GfcUfgCfcCfcUfgUfaGfgUfuGfcUfdTsdT 505aGfcAfaCfcUfaCfaGfgGfgCfaGfcdTsdT AD-46830.1 433AGGUUGCUUAAAAGGGACAdTsdT 506 UGUCCCUUDUAAGCAACCUdTsdT AD-47360.1 434AfgGfuUfgCfuUfaAfaAfgGfgAfcAfdTsdT 507 uGfuCfcCfuUfuUfaAfgCfaAfcCfudTsdTAD-47387.1 435 AGGuuGcuuAAAAGGGAcAdTsdT 508 UGUCCCuUuUAAGCAAccudTsdTAD-46833.1 436 UGCUUAAAAGGGACAGUAUdTsdT 509 AUACUGUCCCUUUUAAGCAdTsdTAD-47337.1 437 UfgCfuUfaAfaAfgGfgAfcAfgUfaUfdTsdT 510aUfaCfuGfuCfcCfuUfuUfaAfgCfadTsdT AD-47364.1 438uGcuuAAAAGGGAcAGuAudTsdT 511 AuACuGUCCCuuuuAAgcadTsdT AD-46813.1 439GCUUAAAAGGGACAGUAUUdTsdT 512 AAUACUGUCCCUUCUAAGCdTsdT AD-47341.1 440GfcUfuAfaAfaGfgGfaCfaGfuAfuUfdTsdT 513 aAfuAfcUfgUfcCfcUfuUfuAfaGfcdTsdTAD-47368.1 441 GcuuAAAAGGGAcAGuAuudTsdT 514 AAuACuGUCCCuuuuAagcdTsdTAD-46817.1 442 CUGGACAAGAAGCUGCUAUdTsdT 515 AUAGCAGCUUCUUGUCCAGdTsdTAD-47345.1 443 CfuGfgAfcAfaGfaAfgCfuGfcUfaUfdTsdT 516aUfaGfcAfgCfuUfcUfuGfuCfcAfgdTsdT AD-47372.1 444cuGGAcAAGAAGcuGcuAudTsdT 517 AuAGCAGCuUCuuGuCcagdTsdT AD-47343.1 445UfcCfcUfgAfaAfgAfcUfaCfuGfgAfdTsdT 518 uCfcAfgUfaGfuCfuUfuCfaGfgGfadTsdTAD-47350.1 446 AfgAfcUfaCfuGfgAfgCfaCfcGfuUfdTsdT 519aAfcGfgUfgCfuCfcAfgUfaGfuCfudTsdT AD-47359.1 447GfgCfuGfcCfuGfaGfaCfcCfcAfaUfdTsdT 520 aUfuGfaGfgUfcCfcAfgGfcAfgCfcdTsdTAD-47386.1 448 GGcuGccuGAGAccucAAudTsdT 521 AuUGAGGUCuCAGGCAgccdTsdTAD-47351.1 449 CfaGfgGfcUfgCfcCfcUfgUfaGfgUfdTsdT 522aCfcUfaCfaGfgGfgCfaGfcCfcUfgdTsdT AD-47378.1 450cAGGGcuGccccuGuAGGudTsdT 523 ACCuACAGGGGCAGCCcugdTsdT AD-47357.1 451CfcCfuGfuAfgGfuUfgCfuUfaAfaAfdTsdT 524 uUfuUfaAfgCfaAfcCfuAfcAfgGfgdTsdTAD-47357.1 452 CfcCfuGfuAfgGfuUfgCfuUfaAfaAfdTsdT 525uUfuUfaAfgCfaAfcCfuAfcAfgGfgdTsdT

TABLE 8 ApoC3 modified siRNA (second set) Activity in cells 10 nM 0.1 nMduplexName 10 nM 0.1 nM SD SD AD-46822.1 0.15 0.31 0.00 0.00 AD-47334.10.41 0.82 0.12 0.10 AD-47361.1 0.89 1.00 0.19 0.03 AD-46825.1 0.13 0.260.01 0.01 AD-47338.1 0.38 0.73 0.12 0.12 AD-47365.1 0.74 0.93 0.01 0.07AD-46828.1 0.15 0.57 0.00 0.04 AD-47342.1 0.38 0.91 0.26 0.06 AD-47369.11.10 1.26 0.18 0.29 AD-46831.1 0.01 0.37 0.00 0.05 AD-47346.1 0.57 0.950.17 0.08 AD-47373.1 0.80 1.06 0.06 0.15 AD-46811.1 0.03 0.31 0.01 0.04AD-47349.1 0.03 0.29 0.02 0.18 AD-47376.1 0.38 0.95 0.15 0.01 AD-46815.10.06 0.37 0.00 0.00 AD-47352.1 0.04 0.35 0.04 0.23 AD-47379.1 0.81 1.030.12 0.05 AD-46818.1 0.03 0.26 0.00 0.03 AD-47355.1 0.23 0.60 0.14 0.08AD-47382.1 0.40 0.81 0.11 0.09 AD-46820.1 0.03 0.38 0.00 0.02 AD-47358.10.05 0.45 0.03 0.31 AD-47385.1 0.53 0.84 0.12 0.19 AD-46823.1 0.02 0.220.00 0.04 AD-47335.1 0.15 0.70 0.05 0.00 AD-47362.1 0.66 1.07 0.01 0.09AD-46826.1 0.02 0.18 0.00 0.02 AD-47339.1 0.19 0.62 0.12 0.04 AD-47366.10.60 0.82 0.02 0.09 AD-46829.1 0.02 0.22 0.01 0.01 AD-47347.1 0.16 0.660.14 0.11 AD-47374.1 0.90 1.15 0.03 0.03 AD-46832.1 0.09 0.56 0.02 0.01AD-47353.1 0.21 0.65 0.04 0.02 AD-47380.1 0.66 1.02 0.07 0.02 AD-46812.10.01 0.10 0.00 0.00 AD-47356.1 0.02 0.13 0.01 0.13 AD-47383.1 0.03 0.210.02 0.10 AD-46816.1 0.04 0.53 0.00 0.01 AD-47336.1 0.10 0.37 0.06 0.14AD-47363.1 0.54 0.88 0.03 0.14 AD-46819.1 0.06 0.49 0.01 0.02 AD-47340.10.20 0.72 0.18 0.21 AD-47367.1 0.99 1.10 0.17 0.07 AD-46821.1 0.19 0.670.01 0.01 AD-47344.1 0.48 0.90 0.16 0.06 AD-47371.1 1.01 0.92 0.14 0.12AD-46824.1 0.02 0.21 0.00 0.02 AD-47348.1 0.19 0.66 0.20 0.24 AD-47375.10.83 0.94 0.06 0.00 AD-46827.1 0.05 0.54 0.01 0.06 AD-47354.1 0.23 0.790.15 0.19 AD-46830.1 0.64 1.01 0.03 0.00 AD-47360.1 0.76 1.22 0.09 0.22AD-47387.1 0.90 0.84 0.04 0.09 AD-46833.1 0.06 0.46 0.01 0.05 AD-47337.10.05 0.23 0.03 0.16 AD-47364.1 0.52 0.79 0.10 0.02 AD-46813.1 0.02 0.270.00 0.01 AD-47341.1 0.04 0.17 0.02 0.11 AD-47368.1 0.46 0.56 0.02 0.13AD-46817.1 0.10 0.29 0.01 0.04 AD-47345.1 0.27 0.58 0.17 0.19 AD-47372.10.34 0.44 0.17 0.04 AD-47343.1 0.21 0.66 0.15 0.18 AD-47350.1 0.12 0.580.05 0.31 AD-47359.1 0.82 1.04 0.10 0.18 AD-47386.1 0.88 1.28 0.15 0.21AD-47351.1 0.48 1.01 0.18 0.14 AD-47378.1 1.10 1.08 0.09 0.03 AD-47357.10.09 0.28 0.02 0.25 AD-47357.1 0.09 0.28 0.02 0.25

TABLE 9 ApoC3 siRNA (third set) duplex names and modification patternsof modified siRNA DuplexName Chemistry AD-45101.1end EndolightAD-45159.1 Fluorolight AD-46822.1 UMdTsdT AD-47334.1 FOME AD-47361.1DECAF AD-45107.1end Endolight AD-45123.1 Fluorolight AD-46825.1 UMdTsdTAD-47338.1 FOME AD-47365.1 DECAF AD-45113.1end Endolight AD-45129.1Fluorolight AD-46828.1 UMdTsdT AD-47342.1 FOME AD-47369.1 DECAFAD-45119.1end Endolight AD-45135.1 Fluorolight AD-46831.1 UMdTsdTAD-47346.1 FOME AD-47373.1 DECAF AD-45078.1end Endolight AD-45140.1Fluorolight AD-46811.1 UMdTsdT AD-47349.1 FOME AD-47376.1 DECAFAD-45084.1end Endolight AD-45145.1 Fluorolight AD-46815.1 UMdTsdTAD-47352.1 FOME AD-47379.1 DECAF AD-45090.1end Endolight AD-45150.1Fluorolight AD-46818.1 UMdTsdT AD-47355.1 FOME AD-47382.1 DECAFAD-45096.1end Endolight AD-45155.1 Fluorolight AD-46820.1 UMdTsdTAD-47358.1 FOME AD-47385.1 DECAF AD-45102.1end Endolight AD-45160.1Fluorolight AD-46823.1 UMdTsdT AD-47335.1 FOME AD-47362.1 DECAFAD-45108.1end Endolight AD-45124.1 Fluorolight AD-46826.1 UMdTsdTAD-47339.1 FOME AD-47366.1 DECAF AD-45120.1end Endolight AD-45136.1Fluorolight AD-46829.1 UMdTsdT AD-47347.1 FOME AD-47374.1 DECAFAD-45127.1end Endolight AD-45146.1 Fluorolight AD-46832.1 UMdTsdTAD-47353.1 FOME AD-47380.1 DECAF AD-45133.1end Endolight AD-45151.1Fluorolight AD-46812.1 UMdTsdT AD-47356.1 FOME AD-47383.1 DECAFAD-45143.1end Endolight AD-45161.1 Fluorolight AD-46816.1 UMdTsdTAD-47336.1 FOME AD-47363.1 DECAF AD-45148.1end Endolight AD-45125.1Fluorolight AD-46819.1 UMdTsdT AD-47340.1 FOME AD-47367.1 DECAFAD-45153.1end Endolight AD-45131.1 Fluorolight AD-46821.1 UMdTsdTAD-47344.1 FOME AD-47371.1 DECAF AD-45158.1end Endolight AD-45137.1Fluorolight AD-46824.1 UMdTsdT AD-47348.1 FOME AD-47375.1 DECAFAD-45128.1end Endolight AD-45147.1 Fluorolight AD-46827.1 UMdTsdTAD-47354.1 FOME AD-45139.1end Endolight AD-45157.1 FluorolightAD-46830.1 UMdTsdT AD-47360.1 FOME AD-47387.1 DECAF AD-45144.1endEndolight AD-45162.1 Fluorolight AD-46833.1 UMdTsdT AD-47337.1 FOMEAD-47364.1 DECAF AD-45149.1end Endolight AD-45126.1 FluorolightAD-46813.1 UMdTsdT AD-47341.1 FOME AD-47368.1 DECAF AD-45154.1endEndolight AD-45132.1 Fluorolight AD-46817.1 UMdTsdT AD-47345.1 FOMEAD-47372.1 DECAF AD-45114.1end Endolight AD-45130.1 FluorolightAD-47343.1 FOME AD-45141.1 Fluorolight AD-45121.1end EndolightAD-47350.1 FOME AD-45138.1end Endolight AD-47359.1 FluorolightAD-47386.1 DECAF AD-45122.1end Endolight AD-47351.1 FOMe AD-47378.1DECAF AD-45152.1 Fluorolight AD-47357.1 FOMe AD-47384.1 DECAF

TABLE 10  ApoC3 modified siRNA (third set) sequences SEQ SEQ ID IDDuplexName NO: Sense Sequence NO: Antisense sequence AD-45101.1end 526uGcAGccccGGGuAcuccudTsdT 650 AGGAGuACCCGGGGCUGcAdTsdT AD-45159.1 527UfGCfAGCfCfCfCfGGGUfACfUfCfCfUfdTsdT 651 AGGAGUfACCCGGGGCUGCfAdTsdTAD-46822.1 528 UGCAGCCCCGGGUACUCCUdTsdT 652 AGGAGUACCCGGGGCUGCAdTsdTAD-47334.1 529 UfgCfaGfcCfcCfgGfgUfaCfuCfcUfdTsdT 653aGfgAfgUfaCfcCfgGfgGfcUfgC fadTsdT AD-47361.1 530uGcAGccccGGGuAcuccudTsdT 654 AGGAGuACCCGGGGCugcadTsdT AD-45107.1end 531GcAGccccGGGuAcuccuudTsdT 655 AAGGAGuACCCGGGGCUGCdTsdT AD-45123.1 532GCfAGCfCfCfCfGGGUfACfUfCfCfUfUfdTsdT 656 AAGGAGUfACCCGGGGCUGCdTsdTAD-46825.1 533 GCAGCCCCGGGUACUCCUUdTsdT 657 AAGGAGUACCCGGGGCUGCdTsdTAD-47338.1 534 GfcAfgCfcCfcGfgGfuAfcUfcCfuUfdTsdT 658aAfgGfaGfuAfcCfcGfgGfgCfuG fcdTsdT AD-47365.1 535GcAGccccGGGuAcuccuudTsdT 659 AAGGAGuACCCGGGGCugcdTsdT AD-45113.1end 536cAAGAccGccAAGGAuGcAdTsdT 660 UGcAUCCUUGGCGGUCUUGdTsdT AD-45129.1 537CfAAGACfCfGCfCfAAGGAUfGCfAdTsdT 661 UGCfAUCCUUGGCGGUCUUGdTsdT AD-46828.1538 CAAGACCGCCAAGGAUGCAdTsdT 662 UGCAUCCUUGGCGGUCUUGdTsdT AD-47342.1 539CfaAfgAfcCfgCfcAfaGfgAfuGfcAfdTsdT 663 uGfcAfuCfcUfuGfgCfgGfuCfuUfgdTsdT AD-47369.1 540 cAAGAccGccAAGGAuGcAdTsdT 664UGCAUCCuUGGCGGuCuugdTsdT AD-45119.1end 541 uGGGuGAccGAuGGcuucAdTsdT 665UGAAGCcAUCGGUcACCcAdTsdT AD-45135.1 542UfGGGUfGACfCfGAUfGGCfUfUfCfAdTsdT 666 UGAAGCCfAUCGGUCfACCCfAdTsdTAD-46831.1 543 UGGGUGACCGAUGGCUUCAdTsdT 667 UGAAGCCAUCGGUCACCCAdTsdTAD-47346.1 544 UfgGfgUfgAfcCfgAfuGfgCfuUfcAfdTsdT 668uGfaAfgCfcAfuCfgGfuCfaCfcC fadTsdT AD-47373.1 545uGGGuGAccGAuGGcuucAdTsdT 669 UGAAGCCAUCGGuCACccadTsdT AD-45078.1end  546GGuGAccGAuGGcuucAGudTsdT 670 ACUGAAGCcAUCGGUcACCdTsdT AD-45140.1 547GGUfGACfCfGAUfGGCfUfUfCfAGUfdTsdT 671 ACUGAAGCCfAUCGGUCfACCdTsdTAD-46811.1 548 GGUGACCGAUGGCUUCAGUdTsdT 672 ACUGAAGCCAUCGGUCACCdTsdTAD-47349.1 549 GfgUfgAfcCfgAfuGfgCfuUfcAfgUfdTsdT 673aCfuGfaAfgCfcAfuCfgGfuCfaC fcdTsdT AD-47376.1 550GGuGAccGAuGGcuucAGudTsdT 674 ACuGAAGCCAuCGGuCaccdTsdT AD-45084.1end 551ccGAuGGcuucAGuucccudTsdT 675 AGGGAACUGAAGCcAUCGGdTsdT AD-45145.1 552CfCfGAUfGGCfUfUfCfAGUfUfCfCfCfUfdTsdT 676 AGGGAACUGAAGCCfAUCGGdTsdTAD-46815.1 553 CCGAUGGCUUCAGUUCCCUdTsdT 677 AGGGAACUGAAGCCAUCGGdTsdTAD-47352.1 554 CfcGfaUfgGfcUfuCfaGfuUfcCfcUfdTsdT 678aGfgGfaAfcUfgAfaGfcCfaUfcG fgdTsdT AD-47379.1 555ccGAuGGcuucAGuucccudTsdT 679 AGGGAACuGAAGCCAucggdTsdT AD-45090.1end 556GAuGGcuucAGuucccuGAdTsdT 680 UcAGGGAACUGAAGCcAUCdTsdT AD-45150.1 557GAUfGGCfUfUfCfAGUfUfCfCfCfUfGAdTsdT 681 UCfAGGGAACUGAAGCCfAUCdTsdTAD-46818.1 558 GAUGGCUUCAGUUCCCUGAdTsdT 682 UCAGGGAACUGAAGCCAUCdTsdTAD-47355.1 559 GfaUfgGfcUfuCfaGfuUfcCfcUfgAfdTsdT 683uCfaGfgGfaAfcUfgAfaGfcCfaU fcdTsdT AD-47382.1 560GAuGGcuucAGuucccuGAdTsdT 684 UCAGGGAACuGAAGCCaucdTsdT AD-45096.1end 561AuGGcuucAGuucccuGAAdTsdT 685 UUcAGGGAACUGAAGCcAUdTsdT AD-45155.1 562AUfGGCfUfUfCfAGUfUfCfCfCfUfGAAdTsdT 686 UUCfAGGGAACUGAAGCCfAUdTsdTAD-46820.1 563 AUGGCUUCAGUUCCCUGAAdTsdT 687 UUCAGGGAACUGAAGCCAUdTsdTAD-47358.1 564 AfuGfgCfuUfcAfgUfuCfcCfuGfaAfdTsdT 688uUfcAfgGfgAfaCfuGfaAfgCfcA fudTsdT AD-47385.1 565AuGGcuucAGuucccuGAAdTsdT 689 UUCAGGGAACuGAAGCcaudTsdT AD-45102.1end 566uGGcuucAGuucccuGAAAdTsdT 690 UUUcAGGGAACUGAAGCcAdTsdT AD-45160.1 567UfGGCfUfUfCfAGUfUfCfCfCfUfGAAAdTsdT 691 UUUCfAGGGAACUGAAGCCfAdTsdTAD-46823.1 568 UGGCUUCAGUUCCCUGAAAdTsdT 692 UUUCAGGGAACUGAAGCCAdTsdTAD-47335.1 569 UfgGfcUfuCfaGfuUfcCfcUfgAfaAfdTsdT 693uUfuCfaGfgGfaAfcUfgAfaGfcC fadTsdT AD-47362.1 570uGGcuucAGuucccuGAAAdTsdT 694 UuUCAGGGAACuGAAGccadTsdT AD-45108.1end 571GcuucAGuucccuGAAAGAdTsdT 695 UCUUUcAGGGAACUGAAGCdTsdT AD-45124.1 572GCfUfUfCfAGUfUfCfCfCfUfGAAAGAdTsdT 696 UCUUUCfAGGGAACUGAAGCdTsdTAD-46826.1 573 GCUUCAGUUCCCUGAAAGAdTsdT 697 UCUUUCAGGGAACUGAAGCdTsdTAD-47339.1 574 GfcUfuCfaGfuUfcCfcUfgAfaAfgAfdTsdT 698uCfuUfuCfaGfgGfaAfcUfgAfaG fcdTsdT AD-47366.1 575GcuucAGuucccuGAAAGAdTsdT 699 UCuUUCAGGGAACuGAagcdTsdT AD-45120.1end 576cuGAAAGAcuAcuGGAGcAdTsdT 700 UGCUCcAGuAGUCUUUcAGdTsdT AD-45136.1 577CfUfGAAAGACfUfACfUfGGAGCfAdTsdT 701 UGCUCCfAGUfAGUCUUUCfAGdTsdTAD-46829.1 578 CUGAAAGACUACUGGAGCAdTsdT 702 UGCUCCAGUAGUCUUUCAGdTsdTAD-47347.1 579 CfuGfaAfaGfaCfuAfcUfgGfaGfcAfdTsdT 703uGfcUfcCfaGfuAfgUfcUfuUfcA fgdTsdT AD-47374.1 580cuGAAAGAcuAcuGGAGcAdTsdT 704 UGCUCCAGuAGuCuuucagdTsdT AD-45127.1end 581AGcAccGuuAAGGAcAAGudTsdT 705 ACUUGUCCUuAACGGUGCUdTsdT AD-45146.1 582AGCfACfCfGUfUfAAGGACfAAGUfdTsdT 706 ACUUGUCCUUfAACGGUGCUdTsdT AD-46832.1583 AGCACCGUUAAGGACAAGUdTsdT 707 ACUUGUCCUUAACGGUGCUdTsdT AD-47353.1 584AfgCfaCfcGfuUfaAfgGfaCfaAfgUfdTsdT 708 aCfuUfgUfcCfuUfaAfcGfgUfgCfudTsdT AD-47380.1 585 AGcAccGuuAAGGAcAAGudTsdT 709ACuUGUCCuUAACGGugcudTsdT AD-45133.1end 586 GcAccGuuAAGGAcAAGuudTsdT 710AACUUGUCCUuAACGGUGCdTsdT AD-45151.1 587 GCfACfCfGUfUfAAGGACfAAGUfUfdTsdT711 AACUUGUCCUUfAACGGUGCdTsdT AD-46812.1 588 GCACCGUUAAGGACAAGUUdTsdT712 AACUUGUCCUUAACGGUGCdTsdT AD-47356.1 589GfcAfcCfgUfuAfaGfgAfcAfaGfuUfdTsdT 713 aAfcUfuGfuCfcUfuAfaCfgGfuGfcdTsdT AD-47383.1 590 GcAccGuuAAGGAcAAGuudTsdT 714AACuUGUCCuuAACGGugcdTsdT AD-45143.1end 591 GcuGccuGAGAccucAAuAdTsdT 715uAUUGAGGUCUcAGGcAGCdTsdT AD-45161.1 592GCfUfGCfCfUfGAGACfCfUfCfAAUfAdTsdT 716 UfAUUGAGGUCUCfAGGCfAGCdTsdTAD-46816.1 593 GCUGCCUGAGACCUCAAUAdTsdT 717 UAUUGAGGUCUCAGGCAGCdTsdTAD-47336.1 594 GfcUfgCfcUfgAfgAfcCfuCfaAfuAfdTsdT 718uAfuUfgAfgGfuCfuCfaGfgCfaG fcdTsdT AD-47363.1 595GcuGccuGAGAccucAAuAdTsdT 719 UAuUGAGGUCuCAGGCagcdTsdT AD-45148.1end 596cuGAGAccucAAuAccccAdTsdT 720 UGGGGuAUUGAGGUCUcAGdTsdT AD-45125.1 597CfUfGAGACfCfUfCfAAUfACfCfCfCfAdTsdT 721 UGGGGUfAUUGAGGUCUCfAGdTsdTAD-46819.1 598 CUGAGACCUCAAUACCCCAdTsdT 722 UGGGGUAUUGAGGUCUCAGdTsdTAD-47340.1 599 CfuGfaGfaCfcUfcAfaUfaCfcCfcAfdTsdT 723uGfgGfgUfaUfuGfaGfgUfcUfcA fgdTsdT AD-47367.1 600cuGAGAccucAAuAccccAdTsdT 724 UGGGGuAuUGAGGuCucagdTsdT AD-45153.1end 601uGAGAccucAAuAccccAAdTsdT 725 UUGGGGuAUUGAGGUCUcAdTsdT AD-45131.1 602UfGAGACfCfUfCfAAUfACfCfCfCfAAdTsdT 726 UUGGGGUfAUUGAGGUCUCfAdTsdTAD-46821.1 603 UGAGACCUCAAUACCCCAAdTsdT 727 UUGGGGUAUUGAGGUCUCAdTsdTAD-47344.1 604 UfgAfgAfcCfuCfaAfuAfcCfcCfaAfdTsdT 728uUfgGfgGfuAfuUfgAfgGfuCfuC fadTsdT AD-47371.1 605uGAGAccucAAuAccccAAdTsdT 729 UuGGGGuAuUGAGGuCucadTsdT AD-45158.1end 606ccucAAuAccccAAGuccAdTsdT 730 UGGACUUGGGGuAUUGAGGdTsdT AD-45137.1 607CfCfUfCfAAUfACfCfCfCfAAGUfCfCfAdTsdT 731 UGGACUUGGGGUfAUUGAGGdTsdTAD-46824.1 608 CCUCAAUACCCCAAGUCCAdTsdT 732 UGGACUUGGGGUAUUGAGGdTsdTAD-47348.1 609 CfcUfcAfaUfaCfcCfcAfaGfuCfcAfdTsdT 733uGfgAfcUfuGfgGfgUfaUfuGfaG fgdTsdT AD-47375.1 610ccucAAuAccccAAGuccAdTsdT 734 UGGACuUGGGGuAuuGaggdTsdT AD-45128.1end 611GcuGccccuGuAGGuuGcudTsdT 735 AGcAACCuAcAGGGGcAGCdTsdT AD-45147.1 612GCfUfGCfCfCfCfUfGUfAGGUfUfGCfUfdTsdT 736 AGCfAACCUfACfAGGGGCfAGCdTsdTAD-46827.1 613 GCUGCCCCUGUAGGUUGCUdTsdT 737 AGCAACCUACAGGGGCAGCdTsdTAD-47354.1 614 GfcUfgCfcCfcUfgUfaGfgUfuGfcUfdTsdT 738aGfcAfaCfcUfaCfaGfgGfgCfaG fcdTsdT AD-45139.1end 615AGGuuGcuuAAAAGGGAcAdTsdT 739 UGUCCCUUUuAAGcAACCUdTsdT AD-45157.1 616AGGUfUfGCfUfUfAAAAGGGACfAdTsdT 740 UGUCCCUUUUfAAGCfAACCUdTsdT AD-46830.1617 AGGUUGCUUAAAAGGGACAdTsdT 741 UGUCCCUUUUAAGCAACCUdTsdT AD-47360.1 618AfgGfuUfgCfuUfaAfaAfgGfgAfcAfdTsdT 742 uGfuCfcCfuUfuUfaAfgCfaAfcCfudTsdT AD-47387.1 619 AGGuuGcuuAAAAGGGAcAdTsdT 743UGUCCCuUuUAAGCAAccudTsdT AD-45144.1end 620 uGcuuAAAAGGGAcAGuAudTsdT 744AuACUGUCCCUUUuAAGcAdTsdT AD-45162.1 621 UfGCfUfUfAAAAGGGACfAGUfAUfdTsdT745 AUfACUGUCCCUUUUfAAGCfAdTsdT AD-46833.1 622 UGCUUAAAAGGGACAGUAUdTsdT746 AUACUGUCCCUUDUAAGCAdTsdT AD-47337.1 623UfgCfuUfaAfaAfgGfgAfcAfgUfaUfdTsdT 747 aUfaCfuGfuCfcCfuUfuUfaAfgCfadTsdT AD-47364.1 624 uGcuuAAAAGGGAcAGuAudTsdT 748AuACuGUCCCuuuuAAgcadTsdT AD-45149.1end 625 GcuuAAAAGGGAcAGuAuudTsdT 749AAuACUGUCCCUUUuAAGCdTsdT AD-45126.1 626 GCfUfUfAAAAGGGACfAGUfAUfUfdTsdT750 AAUfACUGUCCCUUUUfAAGCdTsdT AD-46813.1 627 GCUUAAAAGGGACAGUAUUdTsdT751 AAUACUGUCCCUUUUAAGCdTsdT AD-47341.1 628GfcUfuAfaAfaGfgGfaCfaGfuAfuUfdTsdT 752 aAfuAfcUfgUfcCfcUfuUfuAfaGfcdTsdT AD-47368.1 629 GcuuAAAAGGGAcAGuAuudTsdT 753AAuACuGUCCCuuuuAagcdTsdT AD-45154.1end 630 cuGGAcAAGAAGcuGcuAudTsdT 754AuAGcAGCUUCUUGUCcAGdTsdT AD-45132.1 631 CfUfGGACfAAGAAGCfUfGCfUfAUfdTsdT755 AUfAGCfAGCUUCUUGUCCfAGdTsdT AD-46817.1 632 CUGGACAAGAAGCUGCUAUdTsdT756 AUAGCAGCUUCUUGUCCAGdTsdT AD-47345.1 633CfuGfgAfcAfaGfaAfgCfuGfcUfaUfdTsdT 757 aUfaGfcAfgCfuUfcUfuGfuCfcAfgdTsdT AD-47372.1 634 cuGGAcAAGAAGcuGcuAudTsdT 758AuAGCAGCuUCuuGuCcagdTsdT AD-45114.1end 635 ucccuGAAAGAcuAcuGGAdTsdT 759UCcAGuAGUCUUUcAGGGAdTsdT AD-45130.1 636UfCfCfCfUfGAAAGACfUfACfUfGGAdTsdT 760 UCCfAGUfAGUCUUUCfAGGGAdTsdTAD-47343.1 637 UfcCfcUfgAfaAfgAfcUfaCfuGfgAfdTsdT 761uCfcAfgUfaGfuCfuUfuCfaGfgG fadTsdT AD-45141.1 638AGACfUfACfUfGGAGCfACfCfGUfUfdTsdT 762 AACGGUGCUCCfAGUfAGUCUdTsdTAD-45121.1end 639 AGAcuAcuGGAGcAccGuudTsdT 763 AACGGUGCUCcAGuAGUCUdTsdTAD-47350.1 640 AfgAfcUfaCfuGfgAfgCfaCfcGfuUfdTsdT 764aAfcGfgUfgCfuCfcAfgUfaGfuC fudTsdT AD-45138.1end 641GGcuGccuGAGAccucAAudTsdT 765 AUUGAGGUCUcAGGcAGCCdTsdT AD-47359.1 642GfgCfuGfcCfuGfaGfaCfcUfcAfaUfdTsdT 766 aUfuGfaGfgUfcUfcAfgGfcAfgCfcdTsdT AD-47386.1 643 GGcuGccuGAGAccucAAudTsdT 767AuUGAGGUCuCAGGCAgccdTsdT AD-45122.1end 644 cAGGGcuGccccuGuAGGudTsdT 768ACCuAcAGGGGcAGCCCUGdTsdT AD-47351.1 645CfaGfgGfcUfgCfcCfcUfgUfaGfgUfdTsdT 769 aCfcUfaCfaGfgGfgCfaGfcCfcUfgdTsdT AD-47378.1 646 cAGGGcuGccccuGuAGGudTsdT 770ACCuACAGGGGCAGCCcugdTsdT AD-45152.1 647CfCfCfUfGUfAGGUfUfGCfUfUfAAAAdTsdT 771 UUUUfAAGCfAACCUfACfAGGGdTsdTAD-47357.1 648 CfcCfuGfuAfgGfuUfgCfuUfaAfaAfdTsdT 772uUfuUfaAfgCfaAfcCfuAfcAfgG fgdTsdT AD-47384.1 649cccuGuAGGuuGcuuAAAAdTsdT 773 UuUuAAGCAACCuACAgggdTsdT

TABLE 11 ApoC3 modified siRNA (third set) Activity in cells DuplexName10 nM 0.1 nM 10 nM SD 0.1 nM SD AD-45101.1end 1.01 1.30 0.22 0.42AD-45159.1 0.12 0.53 0.01 0.03 AD-46822.1 0.15 0.31 0.00 0.00 AD-47334.10.41 0.82 0.12 0.10 AD-47361.1 0.89 1.00 0.19 0.03 AD-45107.1end 0.871.06 0.02 0.08 AD-45123.1 0.15 0.41 0.02 0.02 AD-46825.1 0.13 0.26 0.010.01 AD-47338.1 0.38 0.73 0.12 0.12 AD-47365.1 0.74 0.93 0.01 0.07AD-45113.1end 0.97 1.04 0.20 0.01 AD-45129.1 0.20 0.47 0.07 0.06AD-46828.1 0.15 0.57 0.00 0.04 AD-47342.1 0.38 0.91 0.26 0.06 AD-47369.11.10 1.26 0.18 0.29 AD-45119.1end 0.91 0.87 0.18 0.05 AD-45135.1 0.020.08 0.01 0.02 AD-46831.1 0.01 0.37 0.00 0.05 AD-47346.1 0.57 0.95 0.170.08 AD-47373.1 0.80 1.06 0.06 0.15 AD-45078.1end 0.38 0.78 0.08 0.05AD-45140.1 0.07 0.33 0.02 0.06 AD-46811.1 0.03 0.31 0.01 0.04 AD-47349.10.03 0.29 0.02 0.18 AD-47376.1 0.38 0.95 0.15 0.01 AD-45084.1end 0.600.92 0.03 0.06 AD-45145.1 0.04 0.13 0.01 0.02 AD-46815.1 0.06 0.37 0.000.00 AD-47352.1 0.04 0.35 0.04 0.23 AD-47379.1 0.81 1.03 0.12 0.05AD-45090.1end 0.97 0.86 0.06 0.05 AD-45150.1 0.09 0.28 0.01 0.05AD-46818.1 0.03 0.26 0.00 0.03 AD-47355.1 0.23 0.60 0.14 0.08 AD-47382.10.40 0.81 0.11 0.09 AD-45096.1end 0.47 0.82 0.07 0.26 AD-45155.1 0.600.68 0.11 0.07 AD-46820.1 0.03 0.38 0.00 0.02 AD-47358.1 0.05 0.45 0.030.31 AD-47385.1 0.53 0.84 0.12 0.19 AD-45102.1end 0.05 0.22 0.03 0.03AD-45160.1 0.03 0.11 0.00 0.01 AD-46823.1 0.02 0.22 0.00 0.04 AD-47335.10.15 0.70 0.05 0.00 AD-47362.1 0.66 1.07 0.01 0.09 AD-45108.1end 0.020.12 0.00 0.01 AD-45124.1 0.03 0.07 0.01 0.01 AD-46826.1 0.02 0.18 0.000.02 AD-47339.1 0.19 0.62 0.12 0.04 AD-47366.1 0.60 0.82 0.02 0.09AD-45120.1end 0.03 0.08 0.01 0.03 AD-45136.1 0.04 0.10 0.00 0.01AD-46829.1 0.02 0.22 0.01 0.01 AD-47347.1 0.16 0.66 0.14 0.11 AD-47374.10.90 1.15 0.03 0.03 AD-45127.1end 0.58 0.75 0.02 0.05 AD-45146.1 0.060.21 0.00 0.00 AD-46832.1 0.09 0.56 0.02 0.01 AD-47353.1 0.21 0.65 0.040.02 AD-47380.1 0.66 1.02 0.07 0.02 AD-45133.1end 0.03 0.10 0.01 0.04AD-45151.1 0.03 0.05 0.01 0.00 AD-46812.1 0.01 0.10 0.00 0.00 AD-47356.10.02 0.13 0.01 0.13 AD-47383.1 0.03 0.21 0.02 0.10 AD-45143.1end 0.730.94 0.06 0.09 AD-45161.1 0.59 0.54 0.05 0.06 AD-46816.1 0.04 0.53 0.000.01 AD-47336.1 0.10 0.37 0.06 0.14 AD-47363.1 0.54 0.88 0.03 0.14AD-45148.1end 0.80 0.94 0.06 0.10 AD-45125.1 0.16 0.55 0.02 0.01AD-46819.1 0.06 0.49 0.01 0.02 AD-47340.1 0.20 0.72 0.18 0.21 AD-47367.10.99 1.10 0.17 0.07 AD-45153.1end 0.92 1.02 0.06 0.05 AD-45131.1 0.240.64 0.04 0.00 AD-46821.1 0.19 0.67 0.01 0.01 AD-47344.1 0.48 0.90 0.160.06 AD-47371.1 1.01 0.92 0.14 0.12 AD-45158.1end 0.37 0.78 0.04 0.00AD-45137.1 0.02 0.19 0.00 0.06 AD-46824.1 0.02 0.21 0.00 0.02 AD-47348.10.19 0.66 0.20 0.24 AD-47375.1 0.83 0.94 0.06 0.00 AD-45128.1end 0.970.96 0.18 0.08 AD-45147.1 0.20 0.49 0.01 0.16 AD-46827.1 0.05 0.54 0.010.06 AD-47354.1 0.23 0.79 0.15 0.19 AD-45139.1end 1.19 1.13 0.46 0.26AD-45157.1 0.13 0.29 0.02 0.11 AD-46830.1 0.64 1.01 0.03 0.00 AD-47360.10.76 1.22 0.09 0.22 AD-47387.1 0.90 0.84 0.04 0.09 AD-45144.1end 0.450.95 0.15 0.20 AD-45162.1 0.02 0.06 0.00 0.03 AD-46833.1 0.06 0.46 0.010.05 AD-47337.1 0.05 0.23 0.03 0.16 AD-47364.1 0.52 0.79 0.10 0.02AD-45149.1end 0.03 0.07 0.00 0.03 AD-45126.1 0.03 0.08 0.02 0.02AD-46813.1 0.02 0.27 0.00 0.01 AD-47341.1 0.04 0.17 0.02 0.11 AD-47368.10.46 0.56 0.02 0.13 AD-45154.1end 0.14 0.29 0.00 0.04 AD-45132.1 0.300.52 0.04 0.05 AD-46817.1 0.10 0.29 0.01 0.04 AD-47345.1 0.27 0.58 0.170.19 AD-47372.1 0.34 0.44 0.17 0.04 AD-45114.1end 0.37 0.77 0.07 0.15AD-45130.1 0.05 0.12 0.03 0.00 AD-47343.1 0.21 0.66 0.15 0.18 AD-45141.10.03 0.07 0.00 0.00 AD-45121.1end 0.93 0.94 0.05 0.02 AD-47350.1 0.120.58 0.05 0.31 AD-45138.1end 0.86 1.04 0.06 0.33 AD-47359.1 0.82 1.040.10 0.18 AD-47386.1 0.88 1.28 0.15 0.21 AD-45122.1end 0.92 0.97 0.020.11 AD-47351.1 0.48 1.01 0.18 0.14 AD-47378.1 1.10 1.08 0.09 0.03AD-45152.1 0.04 0.13 0.01 0.02 AD-47357.1 0.09 0.28 0.02 0.25 AD-47384.10.09 0.35 0.01 0.17

NCBI Reference Sequence: NM_000040.1, Homo sapiens Apolipoprotein C-III (APOC3), mRNA SEQ ID NO: 1 1tgctcagttc atccctagag gcagctgctc caggaacaga ggtgccatgc agccccgggt 61actccttgtt gttgccctcc tggcgctcct ggcctctgcc cgagcttcag aggccgagga 121tgcctccctt ctcagcttca tgcagggtta catgaagcac gccaccaaga ccgccaagga 181tgcactgagc agcgtgcagg agtcccaggt ggcccagcag gccaggggct gggtgaccga 241tggcttcagt tccctgaaag actactggag caccgttaag gacaagttct ctgagttctg 301ggatttggac cctgaggtca gaccaacttc agccgtggct gcctgagacc tcaatacccc 361aagtccacct gcctatccat cctgcgagct ccttgggtcc tgcaatctcc agggctgccc 421ctgtaggttg cttaaaaggg acagtattct cagtgctctc ctaccccacc tcatgcctgg 481cccccctcca ggcatgctgg cctcccaata aagctggaca agaagctgct atg

We claim:
 1. A double-stranded ribonucleic acid (dsRNA) for inhibitingexpression of an APOC3 gene, wherein the dsRNA comprises a sense strandconsists of UfCfCfCfUfGAAAGACfUfACfUfGGAdTsdT (SEQ ID NO: 215) and anantisense strand consists of UCCfAGUfAGUCUUUCfAGGGAdTsdT (SEQ IDNO:296), wherein “Cf” “Af” “Uf” and Gf” are 2′-fluoro nucleotides and“dTsdT” is a dT-phosphorothioate-dT linkage.
 2. The dsRNA of claim 1,further comprising a ligand.
 3. The dsRNA of claim 2, wherein the ligandis conjugated to the 3′ end of the sense strand of the dsRNA.
 4. ThedsRNA of claim 1, further comprising at least oneN-Acetyl-Galactosamine.
 5. An isolated cell comprising the dsRNA ofclaim
 1. 6. A vector encoding at least one strand of the dsRNA ofclaim
 1. 7. An isolated cell comprising the vector of claim
 6. 8. Apharmaceutical composition for inhibiting expression of an APOC3 genecomprising the dsRNA of claim
 1. 9. The pharmaceutical composition ofclaim 8, comprising a lipid formulation.
 10. The pharmaceuticalcomposition of claim 8, comprising a lipid formulation comprising MC3.11. A method of inhibiting APOC3 expression in a cell, the methodcomprising: (a) contacting the cell the dsRNA of claim 1; and (b)maintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of an APOC3 gene, therebyinhibiting expression of the APOC3 gene in the cell.
 12. The method ofclaim 11, wherein the APOC3 expression is inhibited by at least 30%. 13.A method of treating a disorder mediated by APOC3 expression comprisingadministering to a human in need of such treatment a therapeuticallyeffective amount of the APOC3 dsRNA of claim
 1. 14. The method of claim13, wherein the disorder is elevated triglyceride levels.
 15. The methodof claim 13, wherein the disorder is triglyceride levels >150 mg/dLor >500 mg/dL.